Anti-trem2 antibodies and related methods

ABSTRACT

Provided herein are anti-TREM2 antibodies and related methods of making and using anti-TREM2 antibodies. Also provided are methods and compositions for enhancing an immune response and/or for the treatment of an immune-related condition in an individual, e.g., cancer, comprising killing, disabling, or depleting non-stimulatory myeloid cells using an anti-TREM2 antibody or antigen binding fragment thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 16/408,322, filed May 9, 2019, allowed, which is a continuation ofInternational Application PCT/US2018/065026, filed Dec. 11, 2018, whichclaims the benefit of U.S. Provisional Patent Applications 62/597,827,filed Dec. 12, 2017, and 62/648,089, filed Mar. 26, 2018, each of whichis hereby incorporated by reference in its entirety, for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Oct. 14, 2019, is namedPII008C2_Sequence_Listing.txt, and is 53,100 bytes in size.

BACKGROUND

Immunity plays a role in preventing tumor outgrowth. A complexmicroenvironment can develop within the lesion, and despite therecruitment of T-cells, there is often no effective control of thedeveloping mass. Understanding the balance between tumor elimination andtumor escape may rely on a comprehension of the differential rolesmyeloid cells play in the tumor microenvironment.

Myeloid populations of the tumor microenvironment prominently includemonocytes and neutrophils (sometimes loosely grouped as myeloid-derivedsuppressor cells), macrophages, and dendritic cells. Althoughintra-tumoral myeloid populations, as a whole, have long been considerednon-stimulatory or suppressive, it has more recently been appreciatedthat not all tumor-infiltrating myeloid cells are made equal.

In normal tissues, many of these myeloid cells are essential for properfunctioning of both innate and adaptive immunity and notably for woundrepair. However in the setting of cancer, a significant excess ofmacrophages and dysfunctional or skewed populations of these and othercell types are commonly described. When considered as an aggregatepopulation defined by single markers, such as CD68 or CD163,“macrophage” infiltration is correlated with worse outcomes in subjectsacross multiple tumor types ((de Visser, Cancer Immunol Immunother,2008; 57:1531-9); (Hanada et al., Int J Urol 2000; 7:263-9); (Yao et al.Clin Cancer Res, 520, 2001; 7:4021-6); (Ruffell et al., PNAS, 523 2012;109:2791-801)). But the phenotypic and functional sub-setting ofmacrophages from the tumor microenvironment is complicated by thesimilarity of macrophages and dendritic cells, and is problematic intumor biology. A morphologic criterion has been often applied to theissue; one approach to try to differentiate dendritic cells frommacrophages was based on a more spikey or dendritic morphology for theformer and more veiled or bulbous morphology for the latter (Bell etal., J Exp Med 555, 1999; 190:1417-26). Other groups are trying todifferentiate on the basis of genetic and cell-surface markers.

There is diversity in the antigen-presenting compartment within tumors,and T-cells can differentiate features of antigen-presenting cells(APC). Because T cells are a major driver of tumor immunity,understanding the exact features of their cognate APCs will beimportant. Myeloid cells are prominent among cells capable of presentingtumor-derived antigens to T-cells and thereby maintaining the latter inan activated state. Antigen presentation occurs within the tumor itselfand likely influences the functions of tumor cytotoxic T-lymphocytes(CTLs). T-cell activation by antigen presenting cells (APC) is animportant component in antigen-specific immune responses and tumor cellkilling. As these myeloid populations represent major T-cell-interactingpartners and antigen-presenting cells for incoming tumor-reactivecytotoxic T lymphocytes, understanding their distinctions may guidetherapeutic avenues.

Related patent applications include: PCT/US2015/052682, filed Sep. 28,2015; and PCT/US2016/054104, filed Sep. 28, 2016; each of which isherein incorporated by reference, in their entirety, for all purposes.

All patents, patent applications, publications, documents, and articlescited herein are incorporated herein by reference in their entireties.

SUMMARY

Described herein is an isolated antibody that binds to human TREM2 (SEQID NO:15) and competes for binding to mouse TREM2 (SEQ ID NO: 17) withthe 37017 antibody (SEQ ID NOs: 31 and 32).

In some embodiments, the antibody comprises a CDR-H1 comprising thesequence set forth in SEQ ID NO: 9, a CDR-H2 comprising the sequence setforth in SEQ ID NO: 10, a CDR-H3 comprising the sequence set forth inSEQ ID NO: 11, a CDR-L1 comprising the sequence set forth in SEQ ID NO:12, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 13, and aCDR-L3 comprising the sequence set forth in SEQ ID NO: 14.

In some embodiments, the antibody is afucosylated and comprises the VHsequence shown in SEQ ID NO: 1; the VL sequence shown in SEQ ID NO: 2;and an active human IgG1 Fc region.

In some embodiments, the antibody comprises all 3 heavy chain CDRs ofthe sequence shown in SEQ ID NO:7 and all 3 light chain CDRs of thesequence shown in SEQ ID NO:8.

In some embodiments, the antibody comprises an A to T substitution atposition 97 of the sequence shown in SEQ ID NO:7; and a K to Rsubstitution at position 98 of the sequence shown in SEQ ID NO:7.

In some embodiments, the antibody comprises the VH sequence shown in SEQID NO: 1, 3, or 5.

In some embodiments, the antibody comprises the VH sequence shown in SEQID NO: 1, 3, or 5 and the VL sequence shown in SEQ ID NO: 2, 4, or 6.

In some embodiments, the antibody comprises the VH sequence shown in SEQID NO: 1.

In some embodiments, the antibody comprises the VH sequence shown in SEQID NO: 1 and the VL sequence shown in SEQ ID NO: 2.

In some embodiments, the antibody is the 37012 antibody.

In some embodiments, the antibody comprises the heavy chain sequenceshown in SEQ ID NO: 25 and the light chain sequence shown in SEQ ID NO:26.

In another aspect, described herein is an n isolated antibody that bindsto human TREM2 (SEQ ID NO:15), wherein the antibody competes for bindingto mouse TREM2 (SEQ ID NO: 17) with the 37017 antibody (SEQ ID NOs: 31and 32); and comprises an active human Fc region.

In some embodiments, the antibody is human, humanized, or chimeric.

In some embodiments, the antibody is humanized.

In some embodiments, the antibody binds to human TREM2 with a K_(D) ofless than or equal to about 1, 2, 3, 4, or 5×10⁻⁹, as measured bysurface plasmon resonance (SPR) assay.

In some embodiments, the antibody is capable of specifically killing,depleting, or disabling TREM2+ myeloid cells; optionally non-stimulatorymyeloid cells.

In some embodiments, the antibody has antibody-dependent cell-mediatedcytotoxicity (ADCC) activity. In some embodiments, the antibody hasantibody-mediated cellular phagocytosis (ADCP) activity. In someembodiments, the antibody has complement-dependent cytotoxicity (CDC)activity.

In some embodiments, the antibody kills, disables, or depletes myeloidcells via antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-mediated cellular phagocytosis (ADCP) activity, orcomplement-dependent cytotoxicity (CDC).

In some embodiments, the antibody is at least one of: a monoclonalantibody, a neutral antibody, an antagonistic antibody, an agonistantibody, a polyclonal antibody, an IgG1 antibody, an IgG3 antibody, anafucosylated antibody, a bispecific antibody, a human antibody, ahumanized antibody, a chimeric antibody, a full-length antibody, and anantigen binding fragment thereof.

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the antibody is multispecific.

In some embodiments, the antibody is afucosylated.

In some embodiments, the antibody is an antigen-binding fragmentthereof, a Fab, Fab′, F(ab′)₂, Fv, scFv, (scFv)₂, single chain antibodymolecule, dual variable domain antibody, single variable domainantibody, linear antibody, or V domain antibody.

In some embodiments, the antibody comprises a scaffold, optionallywherein the scaffold is Fc, optionally human Fc.

In some embodiments, the antibody comprises a heavy chain constantregion of a class selected from IgG, IgA, IgD, IgE, and IgM.

In some embodiments, the antibody comprises a heavy chain constantregion of the class IgG and a subclass selected from IgG1, IgG2, IgG3,and IgG4.

In some embodiments, the antibody comprises a heavy chain constantregion of IgG1.

In some embodiments, the Fc comprises one or more modifications, whereinthe one or more modifications result in increased half-life, increasedADCC activity, increased ADCP activity, or increased CDC activitycompared with the Fc without the one or more modifications.

In some embodiments, the Fc binds an Fcγ Receptor selected from thegroup consisting of: FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, andFcγRIIIb.

In another aspect, described herein is an isolated antibody for use inthe treatment of a cancer, wherein the cancer is selected from a solidtumor and a hematological tumor.

In another aspect, described herein is an isolated antibody thatcompetes for binding to human TREM2 with an antibody described herein.

In another aspect, described herein is an isolated antibody that bindsthe human TREM2 epitope bound by an antibody described herein.

In another aspect, described herein is an isolated polynucleotide or setof polynucleotides encoding an antibody described herein, a V_(H)thereof, a V_(L) thereof, a light chain thereof, a heavy chain thereof,or an antigen-binding portion thereof; optionally cDNA.

In another aspect, described herein is a vector or set of vectorscomprising a polynucleotide or set of polynucleotides as describedherein.

In another aspect, described herein is a host cell comprising apolynucleotide or set of polynucleotides as described herein or a vectoror set of vectors described herein.

In another aspect, described herein is a method of producing an antibodycomprising expressing the antibody with a host cell described herein andisolating the expressed antibody.

In another aspect, described herein is a pharmaceutical compositioncomprising an antibody described herein and a pharmaceuticallyacceptable excipient.

In another aspect, described herein is a method of treating orpreventing a disease or condition in a subject in need thereof,comprising administering to the subject an effective amount of anantibody or pharmaceutical composition described herein.

In some embodiments, the disease or condition is cancer.

In some embodiments, the antibody binds to the extracellular domain ofTREM2 on TREM2+ myeloid cells, optionally wherein the myeloid cells areintratumoral. In one embodiment, the antibody binds to the extracellulardomain of TREM2 on myeloid cells, wherein the myeloid cells arenon-stimulatory myeloid cells that are CD45⁺, HLA-DR⁺, CD11c⁺, CD14⁺,and BDCA3⁻, wherein the antibody kills, disables, or depletes thenon-stimulatory myeloid cells via ADCC, CDC, and/or ADCP to a level thatis less than the level of non-stimulatory myeloid cells present in thecancer prior to the contacting of the non-stimulatory myeloid cells withthe antibody, wherein the non-stimulatory myeloid cells are present in apopulation of immune cells comprising stimulatory myeloid cells that areCD45⁺, HLA-DR⁺, CD14⁻, CD11c⁺, BDCA1⁻, and BDCA3⁺ and thenon-stimulatory myeloid cells, and wherein the killing, disabling, ordepleting of the non-stimulatory myeloid cells treats the cancer.

In some embodiments, the antibody kills, disables, or depletes myeloidcells via antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-mediated cellular phagocytosis (ADCP) activity, orcomplement-dependent cytotoxicity (CDC). In some embodiments, theantibody has receptor-ligand blocking, agonism, or antagonism activity.

In some embodiments, the subject is human. In some embodiments, thecancer is a solid cancer. In some embodiments, the cancer is a liquidcancer. In some embodiments, the cancer is selected from the groupconsisting of: melanoma, kidney, hepatobiliary, head-neck squamouscarcinoma (HNSC), pancreatic, colon, bladder, glioblastoma, prostate,lung, breast, ovarian, gastric, kidney, bladder, esophageal, renal,melanoma, and mesothelioma. In some embodiments, the cancer is coloncancer or breast cancer.

In some embodiments, the contacting enhances an immune response in thesubject. In some embodiments, the enhanced immune response is anadaptive immune response. In some embodiments, the enhanced immuneresponse is an innate immune response.

In some embodiments, the subject has previously received, isconcurrently receiving, or will subsequently receive an immunotherapy.In some embodiments, the immunotherapy is at least one of: a checkpointinhibitor; a checkpoint inhibitor of T cells; anti-PD1 antibody;anti-PD1 antibody; anti-CTLA4 antibody; adoptive T cell therapy; CAR-Tcell therapy; a dendritic cell vaccine; a monocyte vaccine; an antigenbinding protein that binds both a T cell and an antigen presenting cell;a BiTE dual antigen binding protein; a toll-like receptor ligand; acytokine; a cytotoxic therapy; a chemotherapy; a radiotherapy; a smallmolecule inhibitor; a small molecule agonist; an immunomodulator; and anepigenetic modulator. In some embodiments, the immunotherapy is ananti-PD1 antibody.

In another aspect, described herein are methods of killing, disabling,or depleting TREM2+ myeloid cells of a subject having cancer, comprisingcontacting the myeloid cells with an anti-TREM2 antibody describedherein or the pharmaceutical composition described herein, optionallywherein the myeloid cells are intratumoral.

In some embodiments, the antibody binds to the extracellular domain ofTREM2, wherein the TREM2+ myeloid cells are non-stimulatory myeloidcells that are CD45⁺, HLA-DR⁺, CD11c⁺, CD14⁺, and BDCA3⁻, wherein theantibody kills, disables, or depletes the non-stimulatory myeloid cellsvia ADCC, CDC, and/or ADCP to a level that is less than the level ofnon-stimulatory myeloid cells present in the cancer prior to thecontacting of the non-stimulatory myeloid cells with the antibody,wherein the non-stimulatory myeloid cells are present in a population ofimmune cells comprising stimulatory myeloid cells that are CD45⁺,HLA-DR⁺, CD14⁻, CD11c⁺, BDCA1⁺, and BDCA3⁺ and the non-stimulatorymyeloid cells, wherein the contacting does not substantially kill,disable, or deplete myeloid cells present outside of the cancer and/orstimulatory myeloid cells present in the cancer, and wherein thekilling, disabling, or depleting of the non-stimulatory myeloid cellstreats the cancer by enhancing an immune response to the cancer.

In some embodiments, the antibody kills the myeloid cells by at leastone of ADCC, CDC, and ADCP. In some embodiments, the antibody disablesthe myeloid cells by at least one of ADCC, CDC, and ADCP. In someembodiments, the antibody depletes the myeloid cells by at least one ofADCC, CDC, and ADCP. In some embodiments, the antibody hasantibody-dependent cell-mediated cytotoxicity (ADCC) activity. In someembodiments, the antibody has complement-dependent cytotoxicity (CDC)activity. In some embodiments, the antibody has antibody-mediatedphagocytosis (ADCP) activity. In some embodiments, the antibody hasreceptor-ligand blocking, agonism, or antagonism activity.

In some embodiments, the myeloid cells are stimulatory myeloid cells. Insome embodiments, the myeloid cells are non-stimulatory myeloid cells.In some embodiments, the myeloid cells comprise at least one ofdendritic cells, tumor-associated macrophages (TAMs), neutrophils, ormonocytes. In some embodiments, the myeloid cells are neutrophils. Insome embodiments, the myeloid cells are tumor-associated macrophages. Insome embodiments, the myeloid cells are intratumoral. In someembodiments, the myeloid cells are in a population of immune cellscomprising stimulatory myeloid cells and non-stimulatory myeloid cells.

In some embodiments, the subject is human. In some embodiments, thecancer is a solid cancer. In some embodiments, the cancer is a liquidcancer. In some embodiments, the cancer is selected from the groupconsisting of: melanoma, kidney, hepatobiliary, head-neck squamouscarcinoma (HNSC), pancreatic, colon, bladder, glioblastoma, prostate,lung, breast, ovarian, gastric, kidney, bladder, esophageal, renal,melanoma, and mesothelioma. In some embodiments, the cancer is coloncancer or breast cancer.

In some embodiments, the contacting enhances an immune response in thesubject. In some embodiments, the enhanced immune response is anadaptive immune response. In some embodiments, the enhanced immuneresponse is an innate immune response.

In some embodiments, the subject has previously received, isconcurrently receiving, or will subsequently receive an immunotherapy.In some embodiments, the immunotherapy is at least one of: a checkpointinhibitor; a checkpoint inhibitor of T cells; anti-PD1 antibody;anti-PD1 antibody; anti-CTLA4 antibody; adoptive T cell therapy; CAR-Tcell therapy; a dendritic cell vaccine; a monocyte vaccine; an antigenbinding protein that binds both a T cell and an antigen presenting cell;a BiTE dual antigen binding protein; a toll-like receptor ligand; acytokine; a cytotoxic therapy; a chemotherapy; a radiotherapy; a smallmolecule inhibitor; a small molecule agonist; an immunomodulator; and anepigenetic modulator. In some embodiments, the immunotherapy is ananti-PD1 antibody.

In another aspect, described herein is a method of detecting TREM2 in asubject having or suspected of having a disease or condition, the methodcomprising: (a) receiving a sample from the subject; and (b) detectingthe presence or the level of TF in the sample by contacting the samplewith an antibody described herein.

In some embodiments, the disease or condition is cancer.

In some embodiments a method described herein comprises administering acheckpoint inhibitor, optionally wherein the checkpoint inhibitor is aninhibitor of the PD1:PDL1 axis, optionally wherein the inhibitor is anantibody, and optionally wherein the antibody is an anti-PD1 antibody oran anti-PDL1 antibody.

In another aspect, described herein is a kit comprising an antibody or apharmaceutical composition disclosed herein and instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows anti-TREM2 PI-7012-mediated anti-tumor activity incombination with anti-PD-1 in the CT-26 syngeneic mouse tumor model.Afucosylation of PI-7012 improves anti-tumor activity in combinationwith anti-PD-1. Shown are the average tumor volumes (10 mice/group).FIG. 1B shows anti-TREM2 PI-7012-mediated anti-tumor activity incombination with anti-PD-1 in the CT-26 syngeneic mouse tumor model.Individual tumor volumes for PI-7012 are shown. FIG. 1C shows anti-TREM2PI-7012-mediated anti-tumor activity in combination with anti-PD-1 inthe CT-26 syngeneic mouse tumor model. Individual tumor volumes forafucosvlated-PI-7012 (afuc-PI-7012) are shown.

FIG. 2 No significant body weight loss with combination treatment. Tenmice in each group were treated with indicated antibodies and bodyweight recorded at frequent intervals. The mean body weight for eachgroup was plotted against study days.

FIG. 3. In addition to H&E staining, tissues were also stained formacrophages using anti-CD68. The intracellular marker CD68 has been usedwidely in the literature as a reliable cytochemical marker toimmunostain monocyte/macrophages in inflamed tissues and tumors. In thelung (Panel E), as well as in the other tissues analyzed, no discernablechange in CD68+ macrophage numbers were observed in any of the treatmentgroups compared to the controls, indicating that anti-TREM2-mediateddepletion occurred specifically in the TME.

FIG. 4A shows anti-CD68 staining of FFPE lung tissue from the indicatedtreatment groups. FIG. 4B shows the results of eight to nine fields ofeach section used for quantitation by light microscopy.

FIG. 5A shows TREM2 expression was absent or very low on cells inselected tissues. FIG. 5B shows TREM2 expression was absent or very lowon cells in selected tissues. Shaded histograms are from TREM2KO andopen histograms from wildtype mice. The antibody used for anti-TREM2staining was clone 237920 from R&D Systems.

FIG. 6. Cell surface expression of TREM2 (open histogram) wassignificantly higher on TAMs compared to granulocytic or monocytic MDSCswithin both MC38 and CT26 tumors. Lymphocytes do not express TREM2.Isotype control staining is shown in grey filled histogram.

FIG. 7. Cell surface expression of TREM2 (open histogram) wassignificantly higher on CD14-derived macrophages compared to any PBMCsubset. Human PBMC or macrophages were either surface stained for TREM2(open histogram) or isotype control (grey histogram). PBMC subsets werediscriminated as neutrophils, monocytes, or T cells using apre-validated multicolor FACS panel.

FIG. 8. Cell surface expression of TREM2 (open histogram) wassignificantly higher on TAMs compared to other infiltrates or non-CD45positive cells. Single cell suspensions from human tumor tissues wereeither surface stained for TREM2 (open histogram) or isotype control(grey histogram). Immune and non-immune subsets were discriminated asusing a pre-validated multicolor FACS panel.

FIG. 9A shows anti-TREM2 mAb afuc-PI7012 combined with anti-PD-1 mAbresults in significant anti-tumor activity in the Panc-02 pancreatictumor model. Tumor volumes were tracked over time in female C57BL/6Jmice implanted with Panc-02 tumor cells and treated with the indicatedmAbs. The Y axis represents mean+/−standard deviation of the averagetumor volumes of 10 mice in each group. FIG. 9B shows tumor volumes fromindividual animals treated with isotype control mAb. FIG. 9C shows tumorvolumes from individual animals treated with anti-TREM2 mAb afuc-PI7012.FIG. 9D shows tumor volumes from individual animals treated withanti-PD-1. FIG. 9E shows tumor volumes from individual animals treatedwith anti-TREM2 mAb afuc-PI7012 and anti-PD-1. FIG. 9F shows anti-TREM2mAb afuc-PI7012 combined with anti-PD-1 mAb results in significantanti-tumor activity in the Panc-02 pancreatic tumor model. Statisticalanalyses of the group average tumor volumes on day 32 after implant foreach treatment group is shown.

FIG. 10. Tumor-free BALB/c mice after anti-TREM2 mAb plus anti-PD-1 mAbtreatment were re-challenged three months later with CT26 tumor cells(square symbols). Age-matched treatment naïve mice (round symbols)received equivalent number of CT26 cells and tracked for tumor growthduring the study period. No additional treatment was provided to themice during the study period.

DETAILED DESCRIPTION Definitions

For purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with any document incorporatedherein by reference, the definition set forth shall control.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

For all compositions described herein, and all methods using acomposition described herein, the compositions can either comprise thelisted components or steps, or can “consist essentially of” the listedcomponents or steps. When a composition is described as “consistingessentially of” the listed components, the composition contains thecomponents listed, and may contain other components which do notsubstantially affect the condition being treated, but do not contain anyother components which substantially affect the condition being treatedother than those components expressly listed; or, if the compositiondoes contain extra components other than those listed whichsubstantially affect the condition being treated, the composition doesnot contain a sufficient concentration or amount of the extra componentsto substantially affect the condition being treated. When a method isdescribed as “consisting essentially of” the listed steps, the methodcontains the steps listed, and may contain other steps that do notsubstantially affect the condition being treated, but the method doesnot contain any other steps which substantially affect the conditionbeing treated other than those steps expressly listed. As a non-limitingspecific example, when a composition is described as “consistingessentially of” a component, the composition may additionally containany amount of pharmaceutically acceptable carriers, vehicles, ordiluents and other such components which do not substantially affect thecondition being treated.

The term “optionally” is meant, when used sequentially, to include fromone to all of the enumerated combinations and contemplates allsubcombinations.

An “effective amount” or “therapeutically effective amount” as usedherein refers to an amount of therapeutic compound, such as ananti-TREM2 antigen binding agent or anti-TREM2 antibody, administered toan individual, either as a single dose or as part of a series of doses,which is effective to produce or contribute to a desired therapeuticeffect, either alone or in combination with another therapeuticmodality. Examples of a desired therapeutic effect is enhancing animmune response, slowing or delaying tumor development; stabilization ofdisease; amelioration of one or more symptoms. An effective amount maybe given in one or more dosages.

The term “treating” as used herein, refers to retarding or reversing theprogress of a condition, such as cancer. The term “treatment,” as usedherein, refers to the act of treating a condition, such as cancer.

An “individual” or “subject” as used herein refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sport, or pet animals, such as dogs, horses, rabbits, cattle, pigs,hamsters, gerbils, mice, ferrets, rats, cats, and the like. In someembodiments, the individual is human. In some embodiments, theindividual is mouse.

The terms “modulate” and “modulation” refer to reducing or inhibitingor, alternatively, activating or increasing, a recited variable.

The terms “increase” and “activate” refer to an increase of 10%, 20%,30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold,4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in arecited variable.

The terms “reduce” and “inhibit” refer to a decrease of 10%, 20%, 30%,40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recitedvariable.

The term “agonize” refers to the activation of receptor signaling toinduce a biological response associated with activation of the receptor.An “agonist” is an entity that binds to and agonizes a receptor.

The term “antagonize” refers to the inhibition of receptor signaling toinhibit a biological response associated with activation of thereceptor. An “antagonist” is an entity that binds to and antagonizes areceptor.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. An exemplary error range is plus or minus 5%. Reference to“about” a value or parameter herein includes (and describes) embodimentsthat are directed to that value or parameter per se.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

For any of the structural and functional characteristics describedherein, methods of determining these characteristics are known in theart.

Antibodies

Structure

The present application provides antibodies and compositions comprisingan antibody which binds a TREM2 protein including antibodies thatdisable non-stimulatory myeloid cells.

The term “antibody” is used herein in its broadest sense and includescertain types of immunoglobulin molecules comprising one or moreantigen-binding domains that specifically bind to an antigen or epitope.An antibody specifically includes intact antibodies (e.g., intactimmunoglobulins), antibody fragments, and multi-specific antibodies.

The recognized immunoglobulin genes include the kappa, lambda, alpha,gamma, delta, epsilon and mu constant region genes, as well as themyriad immunoglobulin variable region genes. Light chains are classifiedas either kappa or lambda. The “class” of an antibody or immunoglobulinrefers to the type of constant domain or constant region possessed byits heavy chain. There are five major classes of antibodies: IgA, IgD,IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG₂, IgG₃, IgG₄, IgA1, and IgA₂. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively.

An exemplary immunoglobulin (antibody) structural unit is composed oftwo pairs of polypeptide chains, each pair having one “light” (about 25kD) and one “heavy” chain (about 50-70 kD). The N-terminal domain ofeach chain defines a variable region of about 100 to 110 or more aminoacids primarily responsible for antigen recognition. The terms variablelight chain (VL) and variable heavy chain (VH) refer to these light andheavy chain domains respectively. The IgG1 heavy chain comprises of theVH, CH1, CH2 and CH3 domains respectively from the N to C-terminus. Thelight chain comprises of the VL and CL domains from N to C terminus. TheIgG1 heavy chain comprises a hinge between the CH1 and CH2 domains. Incertain embodiments, the immunoglobulin constructs comprise at least oneimmunoglobulin domain from IgG, IgM, IgA, IgD, or IgE connected to atherapeutic polypeptide. In some embodiments, the immunoglobulin domainfound in an antibody provided herein, is from or derived from animmunoglobulin based construct such as a diabody, or a nanobody. Incertain embodiments, the immunoglobulin constructs described hereincomprise at least one immunoglobulin domain from a heavy chain antibodysuch as a camelid antibody. In certain embodiments, the immunoglobulinconstructs provided herein comprise at least one immunoglobulin domainfrom a mammalian antibody such as a bovine antibody, a human antibody, acamelid antibody, a mouse antibody or any chimeric antibody.

In some embodiments, the antibodies provided herein comprise a heavychain. In one embodiment, the heavy chain is an IgA. In one embodiment,the heavy chain is an IgD. In one embodiment, the heavy chain is an IgE.In one embodiment, the heavy chain is an IgG. In one embodiment, theheavy chain is an IgM. In one embodiment, the heavy chain is an IgG1. Inone embodiment, the heavy chain is an IgG2. In one embodiment, the heavychain is an IgG3. In one embodiment, the heavy chain is an IgG4. In oneembodiment, the heavy chain is an IgA1. In one embodiment, the heavychain is an IgA2.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe complementarity determining regions (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. Hypervariable regions (HVRs)are also referred to as “complementarity determining regions” (CDRs),and these terms are used herein interchangeably in reference to portionsof the variable region that form the antigen-binding regions. Thisparticular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, Sequences of Proteins of ImmunologicalInterest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987),where the definitions include overlapping or subsets of amino acidresidues when compared against each other. Nevertheless, application ofeither definition to refer to a CDR of an antibody or variants thereofis intended to be within the scope of the term as defined and usedherein. The exact residue numbers which encompass a particular CDR willvary depending on the sequence and size of the CDR. Those skilled in theart can routinely determine which residues comprise a particular CDRgiven the variable region amino acid sequence of the antibody.

The amino acid sequence boundaries of a CDR can be determined by one ofskill in the art using any of a number of known numbering schemes,including those described by Kabat et al., supra (“Kabat” numberingscheme); Al-Lazikani et al., 1997. J. Mol. Biol., 273:927-948 (“Chothia”numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745(“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003,27:55-77 (“IMGT” numbering scheme); and Honegge and Plückthun, J. Mol.Biol., 2001, 309:657-70 (“AHo” numbering scheme); each of which isincorporated by reference in its entirety.

Table A provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1,CDR-H2, and CDR-H3 as identified by the Kabat and Chothia schemes. ForCDR-H1, residue numbering is provided using both the Kabat and Chothianumbering schemes.

CDRs may be assigned, for example, using antibody numbering software,such as Abnum, available at www.bioinf.org.uk/abs/abnum, and describedin Abhinandan and Martin, Immunology, 2008, 45:3832-3839, incorporatedby reference in its entirety.

TABLE A Residues in CDRs according to Kabat and Chothia numberingschemes. CDR Kabat Chothia L1 L24-L34 L24-L34 L2 L50-L56 L50-L56 L3L89-L97 L89-L97 H1 (Kabat Numbering) H31-H35B H26-H32 or H34* H1(Chothia Numbering) H31-H35 H26-H32 H2 H50-H65 H52-H56 H3 H95-H102H95-H102 *The C-terminus of CDR-H1, when numbered using the Kabatnumbering convention, varies between H32 and H34, depending on thelength of the CDR.

The “EU numbering scheme” is generally used when referring to a residuein an antibody heavy chain constant region (e.g., as reported in Kabatet al., supra). Unless stated otherwise, the EU numbering scheme is usedto refer to residues in antibody heavy chain constant regions describedherein.

As used herein, the term “single-chain” refers to a molecule comprisingamino acid monomers linearly linked by peptide bonds. In a particularsuch embodiment, the C-terminus of the Fab light chain is connected tothe N-terminus of the Fab heavy chain in the single-chain Fab molecule.As described in more detail herein, an scFv has a variable domain oflight chain (VL) connected from its C-terminus to the N-terminal end ofa variable domain of heavy chain (VH) by a polypeptide chain.Alternately the scFv comprises of polypeptide chain where in theC-terminal end of the VH is connected to the N-terminal end of VL by apolypeptide chain.

The “Fab fragment” (also referred to as fragment antigen-binding)contains the constant domain (CL) of the light chain and the firstconstant domain (CH1) of the heavy chain along with the variable domainsVL and VH on the light and heavy chains respectively. The variabledomains comprise the complementarity determining loops (CDR, alsoreferred to as hypervariable region) that are involved inantigen-binding. Fab′ fragments differ from Fab fragments by theaddition of a few residues at the carboxy terminus of the heavy chainCH1 domain including one or more cysteines from the antibody hingeregion.

“F(ab′)₂” fragments contain two Fab′ fragments joined, near the hingeregion, by disulfide bonds. F(ab′)₂ fragments may be generated, forexample, by recombinant methods or by pepsin digestion of an intactantibody. The F(ab′) fragments can be dissociated, for example, bytreatment with b-mercaptoethanol.

“Fv” fragments comprise a non-covalently-linked dimer of one heavy chainvariable domain and one light chain variable domain.

The “Single-chain Fv” or “scFv” includes the VH and VL domains of anantibody, wherein these domains are present in a single polypeptidechain. In one embodiment, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen-binding. For a review of scFvsee Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag. New York, pp. 269-315 (1994).HER2 antibody scFv fragments are described in WO93/16185; U.S. Pat. Nos.5,571,894; and 5,587,458.

“scFv-Fc” fragments comprise an scFv attached to an Fc domain. Forexample, an Fc domain may be attached to the C-terminal of the scFv. TheFc domain may follow the V_(H) or V_(L), depending on the orientation ofthe variable domains in the scFv (i.e., V_(H)-V_(L) or V_(L)-V_(H)). Anysuitable Fc domain known in the art or described herein may be used. Insome cases, the Fc domain comprises an IgG4 Fc domain.

The term “single domain antibody” or “sdAb” refers to a molecule inwhich one variable domain of an antibody specifically binds to anantigen without the presence of the other variable domain. Single domainantibodies, and fragments thereof, are described in Arabi Ghahroudi etal., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends inBiochem. Sci., 2001, 26:230-245, each of which is incorporated byreference in its entirety. Single domain antibodies are also known assdAbs or nanobodies. Sdabs are fairly stable and easy to express asfusion partner with the Fc chain of an antibody (Harmsen M M, De Haard HJ (2007). “Properties, production, and applications of camelidsingle-domain antibody fragments”. Appl. Microbiol Biotechnol. 77(1):13-22).

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a naturally occurring antibodystructure and having heavy chains that comprise an Fc region. Forexample, when used to refer to an IgG molecule, a “full length antibody”is an antibody that comprises two heavy chains and two light chains.

The term “epitope” means a portion of an antigen that specifically bindsto an antibody. Epitopes frequently consist of surface-accessible aminoacid residues and/or sugar side chains and may have specific threedimensional structural characteristics, as well as specific chargecharacteristics. Conformational and non-conformational epitopes aredistinguished in that the binding to the former but not the latter maybe lost in the presence of denaturing solvents. An epitope may compriseamino acid residues that are directly involved in the binding, and otheramino acid residues, which are not directly involved in the binding. Theepitope to which an antibody binds can be determined using knowntechniques for epitope determination such as, for example, testing forantibody binding to TREM2 variants with different point-mutations, or tochimeric TREM2 variants.

A “multispecific antibody” is an antibody that comprises two or moredifferent antigen-binding domains that collectively specifically bindtwo or more different epitopes. The two or more different epitopes maybe epitopes on the same antigen (e.g., a single TREM2 molecule expressedby a cell) or on different antigens (e.g., different TREM2 moleculesexpressed by the same cell, or a TREM2 molecule and a non-TREM2molecule). In some aspects, a multi-specific antibody binds twodifferent epitopes (i.e., a “bispecific antibody”). In some aspects, amulti-specific antibody binds three different epitopes (i.e., a“trispecific antibody”).

A “monospecific antibody” is an antibody that comprises one or morebinding sites that specifically bind to a single epitope. An example ofa monospecific antibody is a naturally occurring IgG molecule which,while divalent (i.e., having two antigen-binding domains), recognizesthe same epitope at each of the two antigen-binding domains. The bindingspecificity may be present in any suitable valency.

The term “monoclonal antibody” refers to an antibody from a populationof substantially homogeneous antibodies. A population of substantiallyhomogeneous antibodies comprises antibodies that are substantiallysimilar and that bind the same epitope(s), except for variants that maynormally arise during production of the monoclonal antibody. Suchvariants are generally present in only minor amounts. A monoclonalantibody is typically obtained by a process that includes the selectionof a single antibody from a plurality of antibodies. For example, theselection process can be the selection of a unique clone from aplurality of clones, such as a pool of hybridoma clones, phage clones,yeast clones, bacterial clones, or other recombinant DNA clones. Theselected antibody can be further altered, for example, to improveaffinity for the target (“affinity maturation”), to humanize theantibody, to improve its production in cell culture, and/or to reduceits immunogenicity in a subject.

“Effector functions” refer to those biological activities mediated bythe Fc region of an antibody, which activities may vary depending on theantibody isotype. Examples of antibody effector functions include C1qbinding to activate complement dependent cytotoxicity (CDC), Fc receptorbinding to activate antibody-dependent cellular cytotoxicity (ADCC), andantibody dependent cellular phagocytosis (ADCP), receptor ligandblocking, agonism, or antagonism.

Anti-TREM2 antibodies can include those described herein such as theclones set forth in the tables. In some embodiments, the antibodycomprises an alternative scaffold. In some embodiments, the antibodyconsists of an alternative scaffold. In some embodiments, the antibodyconsists essentially of an alternative scaffold. In some embodiments,the antibody comprises an antibody fragment. In some embodiments, theantibody consists of an antibody fragment. In some embodiments, theantibody consists essentially of an antibody fragment. A “TREM2antibody,” “anti-TREM2 antibody,” or “TREM2-specific antibody” is anantibody, as provided herein, which specifically binds to the antigenTREM2. In some embodiments, the antibody binds the extracellular domainof TREM2. In certain embodiments, a TREM2 antibody provided herein bindsto an epitope of TREM2 that is conserved between or among TREM2 proteinsfrom different species.

The term “chimeric antibody” refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

“Humanized” forms of non-human antibodies are chimeric antibodies thatcontain minimal sequence derived from the non-human antibody. Ahumanized antibody is generally a human antibody (recipient antibody) inwhich residues from one or more CDRs are replaced by residues from oneor more CDRs of a non-human antibody (donor antibody). The donorantibody can be any suitable non-human antibody, such as a mouse, rat,rabbit, chicken, or non-human primate antibody having a desiredspecificity, affinity, or biological effect. The humanized antibody isless likely to induce an immune response, and/or induces a less severeimmune response, as compared to the non-human species antibody, when itis administered to a human subject. In some instances, selectedframework region residues of the recipient antibody are replaced by thecorresponding framework region residues from the donor antibody.Humanized antibodies may also comprise residues that are not found ineither the recipient antibody or the donor antibody. Such modificationsmay be made to further refine antibody function. Examples of how to makehumanized antibodies can be found in U.S. Pat. Nos. 6,054,297, 5,886,152and 5,877,293, each of which is incorporated by reference in itsentirety. For further details, see Jones et al., Nature, 1986,321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta,Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporatedby reference in its entirety.

In one embodiment, the constant domain(s) from a human antibody arefused to the variable domain(s) of a non-human species. In anotherembodiment, one or more amino acid residues in one or more CDR sequencesof a non-human antibody are changed to reduce the likely immunogenicityof the non-human antibody when it is administered to a human subject,wherein the changed amino acid residues either are not critical forimmunospecific binding of the antibody to its antigen, or the changes tothe amino acid sequence that are made are conservative changes, suchthat the binding of the humanized antibody to the antigen is notsignificantly worse than the binding of the non-human antibody to theantigen.

A “human antibody” is one which possesses an amino acid sequencecorresponding to that of an antibody produced by a human or a humancell, or derived from a non-human source that utilizes a human antibodyrepertoire or human antibody-encoding sequences (e.g., obtained fromhuman sources or designed de novo). Human antibodies specificallyexclude humanized antibodies. In one embodiment, all of the variable andconstant domains are derived from human immunoglobulin sequences (afully human antibody). These antibodies may be prepared in a variety ofways including through the immunization with an antigen of interest of amouse that is genetically modified to express antibodies derived fromhuman heavy and/or light chain-encoding genes.

In some embodiments, the antibodies provided herein comprise an antibodyfragment. In some embodiments, the antibodies provided herein consist ofan antibody fragment. In some embodiments, the antibodies providedherein consist essentially of an antibody fragment. In some embodiments,the antibody fragment is an Fv fragment. In some embodiments, theantibody fragment is a Fab fragment. In some embodiments, the antibodyfragment is a F(ab′)₂ fragment. In some embodiments, the antibodyfragment is a Fab′ fragment. In some embodiments, the antibody fragmentis an scFv (sFv) fragment. In some embodiments, the antibody fragment isan scFv-Fc fragment. In some embodiments, the antibody fragment is afragment of a single domain antibody.

Sequences of TREM2 Antibodies

V_(H) Domains

In some embodiments, an antibody provided herein comprises a V_(H)sequence selected from SEQ ID NOs: 1·3, 5, and 7. In some embodiments,an antibody provided herein comprises a V_(H) sequence of SEQ ID NO: 1.In some embodiments, an antibody provided herein comprises a V_(H)sequence of SEQ ID NO:3. In some embodiments, an antibody providedherein comprises a V_(H) sequence of SEQ ID NO:5. In some embodiments,an antibody provided herein comprises a V_(H) sequence of SEQ ID NO:7.

In some embodiments, an antibody provided herein comprises a V_(H)sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99%identity to an illustrative V_(H) sequence provided in SEQ ID NOs: 1, 3,5, and 7. In some embodiments, an antibody provided herein comprises aV_(H) sequence provided in SEQ ID NOs: 1, 3, 5, and 7, with up to 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 amino acid substitutions. In some aspects, the amino acidsubstitutions are conservative amino acid substitutions. In someembodiments, the antibodies described in this paragraph are referred toherein as “variants.” In some embodiments, such variants are derivedfrom a sequence provided herein, for example, by affinity maturation,site directed mutagenesis, random mutagenesis, or any other method knownin the art or described herein. In some embodiments, such variants arenot derived from a sequence provided herein and may, for example, beisolated de novo according to the methods provided herein for obtainingantibodies.

V_(L) Domains

In some embodiments, an antibody provided herein comprises a V_(L)sequence selected from SEQ ID NOs: 2, 4, 6, and 8. In some embodiments,an antibody provided herein comprises a V_(L) sequence of SEQ ID NO:2.In some embodiments, an antibody provided herein comprises a V_(L)sequence of SEQ ID NO:4. In some embodiments, an antibody providedherein comprises a V_(L) sequence of SEQ ID NO:6. In some embodiments,an antibody provided herein comprises a V_(L) sequence of SEQ ID NO:8.

In some embodiments, an antibody provided herein comprises a V_(L)sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99%identity to an illustrative V_(L) sequence provided in SEQ ID NOs: 2, 4,6, and 8. In some embodiments, an antibody provided herein comprises aV_(L) sequence provided in SEQ ID NOs: 2, 4, 6, and 8, with up to 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 amino acid substitutions. In some aspects, the amino acidsubstitutions are conservative amino acid substitutions. In someembodiments, the antibodies described in this paragraph are referred toherein as “variants.” In some embodiments, such variants are derivedfrom a sequence provided herein, for example, by affinity maturation,site directed mutagenesis, random mutagenesis, or any other method knownin the art or described herein. In some embodiments, such variants arenot derived from a sequence provided herein and may, for example, beisolated de novo according to the methods provided herein for obtainingantibodies.

V_(H)-V_(L) Combinations

In some embodiments, an antibody provided herein comprises a V_(H)sequence selected from SEQ ID NOs: 1, 3, 5, and 7; and a V_(L) sequenceselected from SEQ ID NOs: 2, 4, 6, and 8.

In some embodiments, an antibody provided herein comprises a V_(H)sequence of SEQ ID NO:1 and a V_(L) sequence of SEQ ID NO:2. In someembodiments, an antibody provided herein comprises a V_(H) sequence ofSEQ ID NO:3 and a V_(L) sequence of SEQ ID NO:4. In some embodiments, anantibody provided herein comprises a V₁ sequence of SEQ ID NO: 5 and aV_(L) sequence of SEQ ID NO:6. In some embodiments, an antibody providedherein comprises a V_(H) sequence of SEQ ID NO: 7 and a V_(L) sequenceof SEQ ID NO:8. In certain aspects, any of SEQ ID NOs: 1, 3, 5, and 7can be combined with any of SEQ ID NOs: 2, 4, 6, and 8. For example, SEQID NO:1 can be combined with any of SEQ ID NO: 2, 4, 6, or 8. As anotherexample, SEQ ID NO:2 can be combined with any of SEQ ID NO: 1, 3, 5, or7.

In some embodiments, an antibody provided herein comprises a V_(H)sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99%identity to an illustrative V_(H) sequence provided in SEQ ID NOs: 1, 3,5, and 7; and a V_(L) sequence having at least about 50%, 60%, 70%, 80%,90%, 95%, or 99% identity to an illustrative VL sequence provided in SEQID NOs: 2, 4, 6, and 8. In some embodiments, an antibody provided hereincomprises a VH sequence provided in SEQ ID NOs: 1, 3, 5, and 7, with upto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 amino acid substitutions, and a VL sequenceprovided in SEQ ID NOs: 2, 4, 6, and 8, with up to 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25amino acid substitutions. In some aspects, the amino acid substitutionsare conservative amino acid substitutions. In some embodiments, theantibodies described in this paragraph are referred to herein as“variants.” In some embodiments, such variants are derived from asequence provided herein, for example, by affinity maturation, sitedirected mutagenesis, random mutagenesis, or any other method known inthe art or described herein. In some embodiments, such variants are notderived from a sequence provided herein and may, for example, beisolated de novo according to the methods provided herein for obtainingantibodies.

CDRs

In some embodiments, an antibody provided herein comprises one to threeCDRs of a V_(H) domain selected from SEQ ID NOs: 1, 3, 5, and 7. In someembodiments, an antibody provided herein comprises two to three CDRs ofa V_(u) domain selected from SEQ ID NOs: 1, 3, 5, and 7. In someembodiments, an antibody provided herein comprises three CDRs of a VHdomain selected from SEQ ID NOs: 1, 3, 5, and 7. In some aspects, theCDRs are Exemplary CDRs. In some aspects, the CDRs are Kabat CDRs. Insome aspects, the CDRs are Chothia CDRs. In some aspects, the CDRs areAbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects,the CDRs are IMGT CDRs.

In some embodiments, the CDRs are CDRs having at least about 50%, 75%,80%, 85%, 90%, or 95% identity with a CDR-H1, CDR-H2, or CDR-H3 of SEQID NOs: 1, 3, 5, and 7. In some embodiments, the CDR-H1 is a CDR-H1 of aVH domain selected from SEQ ID NOs: 1, 3, 5, and 7, with up to 1, 2, 3,4, or 5 amino acid substitutions. In some embodiments, the CDR-H2 is aCDR-H2 of a VH domain selected from SEQ ID NOs: 1, 3, 5, and 7, with upto 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In someembodiments, the CDR-H3 is a CDR-H3 of a VH domain selected from SEQ IDNOs: 1, 3, 5, and 7, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acidsubstitutions. In some aspects, the amino acid substitutions areconservative amino acid substitutions. In some embodiments, theantibodies described in this paragraph are referred to herein as“variants.” In some embodiments, such variants are derived from asequence provided herein, for example, by affinity maturation, sitedirected mutagenesis, random mutagenesis, or any other method known inthe art or described herein. In some embodiments, such variants are notderived from a sequence provided herein and may, for example, beisolated de novo according to the methods provided herein for obtainingantibodies.

In some embodiments, an antibody provided herein comprises one to threeCDRs of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8. In someembodiments, an antibody provided herein comprises two to three CDRs ofa VL domain selected from SEQ ID NOs: 2, 4, 6, and 8. In someembodiments, an antibody provided herein comprises three CDRs of a VLdomain selected from SEQ ID NOs: 2, 4, 6, and 8. In some aspects, theCDRs are Exemplary CDRs. In some aspects, the CDRs are Kabat CDRs. Insome aspects, the CDRs are Chothia CDRs. In some aspects, the CDRs areAbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects,the CDRs are IMGT CDRs.

In some embodiments, the CDRs are CDRs having at least about 50%, 75%,80%, 85%, 90%, or 95% identity with a CDR-L1, CDR-L2, or CDR-L3 of SEQID NOs: 2, 4, 6, and 8. In some embodiments, the CDR-L1 is a CDR-L1 of aVL domain selected from SEQ ID NOs: 2, 4, 6, and 8, with up to 1, 2, 3,4, or 5 amino acid substitutions. In some embodiments, the CDR-L2 is aCDR-L2 of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8, with upto 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In someembodiments, the CDR-L3 is a CDR-L3 of a VL domain selected from SEQ IDNOs: 2, 4, 6, and 8, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acidsubstitutions. In some aspects, the amino acid substitutions areconservative amino acid substitutions. In some embodiments, theantibodies described in this paragraph are referred to herein as“variants.” In some embodiments, such variants are derived from asequence provided herein, for example, by affinity maturation, sitedirected mutagenesis, random mutagenesis, or any other method known inthe art or described herein. In some embodiments, such variants are notderived from a sequence provided herein and may, for example, beisolated de novo according to the methods provided herein for obtainingantibodies.

In some embodiments, an antibody provided herein comprises one to threeCDRs of a VH domain selected from SEQ ID NOs: 1, 3, 5, and 7 and one tothree CDRs of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8. Insome embodiments, an antibody provided herein comprises two to threeCDRs of a VH domain selected from SEQ ID NOs: 1, 3, 5, and 7 and two tothree CDRs of a VL domain selected from SEQ ID NOs: 2, 4, 6, and 8. Insome embodiments, an antibody provided herein comprises three CDRs of aVH domain selected from SEQ ID NOs: 1, 3, 5, and 7 and three CDRs of aVL domain selected from SEQ ID NOs: 2, 4, 6, and 8. In some aspects, theCDRs are Exemplary CDRs. In some aspects, the CDRs are Kabat CDRs. Insome aspects, the CDRs are Chothia CDRs. In some aspects, the CDRs areAbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects,the CDRs are IMGT CDRs.

In some embodiments, an antibody provided herein comprises a CDR-H3selected of SEQ ID NO: 11. In some aspects, the CDR-H3 has at leastabout 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-H3 of SEQ IDNO: 11. In some embodiments, the CDR-H3 is a CDR-H3 selected of SEQ IDNO: 11, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions.In some aspects, the amino acid substitutions are conservative aminoacid substitutions. In some embodiments, the antibodies described inthis paragraph are referred to herein as “variants.” In someembodiments, such variants are derived from a sequence provided herein,for example, by affinity maturation, site directed mutagenesis, randommutagenesis, or any other method known in the art or described herein.In some embodiments, such variants are not derived from a sequenceprovided herein and may, for example, be isolated de novo according tothe methods provided herein for obtaining antibodies.

In some embodiments, an antibody provided herein comprises a CDR-H2 ofSEQ ID NO: 10. In some aspects, the CDR-H2 has at least about 50%, 75%,80%, 85%, 90%, or 95% identity with a CDR-H2 of SEQ ID NO: 10. In someembodiments, the CDR-H2 is a CDR-H2 of SEQ ID NO: 10, with up to 1, 2,3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the aminoacid substitutions are conservative amino acid substitutions. In someembodiments, the antibodies described in this paragraph are referred toherein as “variants.” In some embodiments, such variants are derivedfrom a sequence provided herein, for example, by affinity maturation,site directed mutagenesis, random mutagenesis, or any other method knownin the art or described herein. In some embodiments, such variants arenot derived from a sequence provided herein and may, for example, beisolated de novo according to the methods provided herein for obtainingantibodies.

In some embodiments, an antibody provided herein comprises a CDR-H1 ofSEQ ID NO: 9. In some aspects, the CDR-H1 has at least about 50%, 75%,80%, 85%, 90%, or 95% identity with a CDR-H1 of SEQ ID NO: 9. In someembodiments, the CDR-H1 is a CDR-H1 of SEQ ID NO: 9, with up to 1, 2, 3,4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the aminoacid substitutions are conservative amino acid substitutions. In someembodiments, the antibodies described in this paragraph are referred toherein as “variants.” In some embodiments, such variants are derivedfrom a sequence provided herein, for example, by affinity maturation,site directed mutagenesis, random mutagenesis, or any other method knownin the art or described herein. In some embodiments, such variants arenot derived from a sequence provided herein and may, for example, beisolated de novo according to the methods provided herein for obtainingantibodies.

In some embodiments, an antibody provided herein comprises a CDR-H3 ofSEQ ID NO: 11 and a CDR-H2 of SEQ ID NO: 10. In some embodiments, anantibody provided herein comprises a CDR-H3 of SEQ ID NO: 11, a CDR-H2of SEQ ID NO: 10, and a CDR-H1 of SEQ ID NO: 9. In some embodiments, theCDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity witha CDR-H3 of SEQ ID NO: 11, the CDR-H2 has at least about 50%, 75%, 80%,85%, 90%, or 95% identity with a CDR-H2 of SEQ ID NO: 10, and the CDR-H1has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with aCDR-H1 of SEQ ID NO: 9. In some embodiments, the CDR-H3 is a CDR-H3 ofSEQ ID NO: 11, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acidsubstitutions; the CDR-H2 is a CDR-H2 of SEQ ID NO: 10, with up to 1, 2,3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is a CDR-H1of SEQ ID NO: 9, with up to 1, 2, 3, 4, or 5 amino acid substitutions.In some aspects, the amino acid substitutions are conservative aminoacid substitutions. In some embodiments, the antibody described in thisparagraph are referred to herein as “variants.” In some embodiments,such variants are derived from a sequence provided herein, for example,by affinity maturation, site directed mutagenesis, random mutagenesis,or any other method known in the art or described herein. In someembodiments, such variants are not derived from a sequence providedherein and may, for example, be isolated de novo according to themethods provided herein for obtaining antibodies.

In some embodiments, an antibody provided herein comprises a CDR-L3 ofSEQ ID NO: 14. In some aspects, the CDR-L3 has at least about 50%, 75%,80%, 85%, 90%, or 95% identity with a CDR-L3 of SEQ ID NO: 14. In someembodiments, the CDR-L3 is a CDR-L3 of SEQ ID NO: 14, with up to 1, 2,3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the aminoacid substitutions are conservative amino acid substitutions. In someembodiments, the antibodies described in this paragraph are referred toherein as “variants.” In some embodiments, such variants are derivedfrom a sequence provided herein, for example, by affinity maturation,site directed mutagenesis, random mutagenesis, or any other method knownin the art or described herein. In some embodiments, such variants arenot derived from a sequence provided herein and may, for example, beisolated de novo according to the methods provided herein for obtainingantibodies.

In some embodiments, an antibody provided herein comprises a CDR-L2 ofSEQ ID NO: 13. In some aspects, the CDR-L2 has at least about 50%, 75%,80%, 85%, 90%, or 95% identity with a CDR-L2 of SEQ ID NO: 13. In someembodiments, the CDR-L2 is a CDR-L2 of SEQ ID NO: 13, with up to 1, 2,3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the aminoacid substitutions are conservative amino acid substitutions. In someembodiments, the antibodies described in this paragraph are referred toherein as “variants.” In some embodiments, such variants are derivedfrom a sequence provided herein, for example, by affinity maturation,site directed mutagenesis, random mutagenesis, or any other method knownin the art or described herein. In some embodiments, such variants arenot derived from a sequence provided herein and may, for example, beisolated de novo according to the methods provided herein for obtainingantibodies.

In some embodiments, an antibody provided herein comprises a CDR-L1 ofSEQ ID NO: 12. In some aspects, the CDR-L1 has at least about 50%, 75%,80%, 85%, 90%, or 95% identity with a CDR-L1 of SEQ ID NO: 12. In someembodiments, the CDR-L1 is a CDR-L1 of SEQ ID NO: 12, with up to 1, 2,3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the aminoacid substitutions are conservative amino acid substitutions. In someembodiments, the antibodies described in this paragraph are referred toherein as “variants.” In some embodiments, such variants are derivedfrom a sequence provided herein, for example, by affinity maturation,site directed mutagenesis, random mutagenesis, or any other method knownin the art or described herein. In some embodiments, such variants arenot derived from a sequence provided herein and may, for example, beisolated de novo according to the methods provided herein for obtainingantibodies.

In some embodiments, an antibody provided herein comprises a CDR-L3 ofSEQ ID NO: 14 and a CDR-L2 of SEQ ID NO: 13. In some embodiments, anantibody provided herein comprises a CDR-L3 of SEQ ID NO: 14, a CDR-L2of SEQ ID NO: 13, and a CDR-L1 of SEQ ID NO: 12. In some embodiments,the CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identitywith a CDR-L3 of SEQ ID NO: 14, the CDR-L2 has at least about 50%, 75%,80%, 85%, 90%, or 95% identity with a CDR-L2 of SEQ ID NO: 13, and theCDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity witha CDR-L1 of SEQ ID NO: 12. In some embodiments, the CDR-L3 is a CDR-L3of SEQ ID NO: 14, with up to 1, 2, 3, 4, or 5 amino acid substitutions;the CDR-L2 is a CDR-L2 of SEQ ID NO: 13, with up to 1, 2, 3, or 4 aminoacid substitutions; and the CDR-L1 is a CDR-L1 of SEQ ID NO: 12, with upto 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, theamino acid substitutions are conservative amino acid substitutions. Insome embodiments, the antibodies described in this paragraph arereferred to herein as “variants.” In some embodiments, such variants arederived from a sequence provided herein, for example, by affinitymaturation, site directed mutagenesis, random mutagenesis, or any othermethod known in the art or described herein. In some embodiments, suchvariants are not derived from a sequence provided herein and may, forexample, be isolated de novo according to the methods provided hereinfor obtaining antibodies.

In some embodiments, an antibody provided herein comprises a CDR-H3 ofSEQ ID NO: 11, a CDR-H2 of SEQ ID NO: 10, a CDR-H1 of SEQ ID NO: 9, aCDR-L3 of SEQ ID NO: 14, a CDR-L2 of SEQ ID NO: 13, and a CDR-L1 of SEQID NO: 12. In some embodiments, the CDR-H3 has at least about 50%, 75%,80%, 85%, 90%, or 95% identity with a CDR-H3 of SEQ ID NO: 11, theCDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity witha CDR-H2 of SEQ ID NO: 10, the CDR-H1 has at least about 50%, 75%, 80%,85%, 90%, or 95% identity with a CDR-H1 of SEQ ID NO: 9, the CDR-L3 hasat least about 50%, 75%, 810%, 85%, 90%, or 95% identity with a CDR-L3of SEQ ID NO: 14, the CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%,or 95% identity with a CDR-L2 of SEQ ID NO: 13, and the CDR-L1 has atleast about 50%, 75%, 80%, 85%, 90%, or 95% identity with a CDR-L1 ofSEQ ID NO: 12. In some embodiments, the CDR-H3 is a CDR-H3 of SEQ ID NO:11, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; theCDR-H2 is a CDR-H2 of SEQ ID NO: 10, with up to 1, 2, 3, 4, 5, 6, 7, or8 amino acid substitutions; the CDR-H1 is a CDR-H1 of SEQ ID NO: 9, withup to 1, 2, 3, 4, or 5 amino acid substitutions; the CDR-L3 is a CDR-L3of SEQ ID NO: 14, with up to 1, 2, 3, 4, or 5 amino acid substitutions;the CDR-L2 is a CDR-L2 of SEQ ID NO: 13, with up to 1, 2, 3, or 4 aminoacid substitutions; and the CDR-L1 is a CDR-L1 of SEQ ID NO: 12, with upto 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, theamino acid substitutions are conservative amino acid substitutions. Insome embodiments, the antibodies described in this paragraph arereferred to herein as “variants.” In some embodiments, such variants arederived from a sequence provided herein, for example, by affinitymaturation, site directed mutagenesis, random mutagenesis, or any othermethod known in the art or described herein. In some embodiments, suchvariants are not derived from a sequence provided herein and may, forexample, be isolated de novo according to the methods provided hereinfor obtaining antibodies.

In some embodiments, an antibody provided herein comprises a CDR-H1 ofSEQ ID NO:9, a CDR-H2 of SEQ ID NO: 10, a CDR-H3 of SEQ ID NO: 11, aCDR-L1 of SEQ ID NO: 12, a CDR-L2 of SEQ ID NO:13, and a CDR-L1 of SEQID NO:14.

Fc Region

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Unless otherwise specifiedherein, numbering of amino acid residues in the Fc region or constantregion is according to the EU numbering system, also called the EUindex, as described in Kabat et al, Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991. An “Fc polypeptide” of adimeric Fc as used herein refers to one of the two polypeptides formingthe dimeric Fc domain, i.e. a polypeptide comprising C-terminal constantregions of an immunoglobulin heavy chain, capable of stableself-association. For example, an Fc polypeptide of a dimeric IgG Fccomprises an IgG CH2 and an IgG CH3 constant domain sequence. An Fc canbe of the class IgA, IgD, IgE, IgG, and IgM, and several of these may befurther divided into subclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃,IgG₄, IgA₁, and IgA₂.

The terms “Fc receptor” and “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. For example, an FcR can be anative sequence human FcR. Generally, an FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Immunoglobulins of other isotypes can alsobe bound by certain FcRs (see, e.g., Janeway et al., Immuno Biology: theimmune system in health and disease, (Elsevier Science Ltd., NY) (4thed., 1999)). Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain (reviewed in DaCron,Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch andKinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41(1995). Other FcRs, including those to be identified in the future, areencompassed by the term “FcR” herein. The term also includes theneonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976);and Kim et al., J. Immunol. 24:249 (1994)).

In some embodiments, an antibody is an IgG1 antibody.

In some embodiments, an antibody is an IgG3 antibody.

In some embodiments, an antibody is an IgG2 antibody.

In some embodiments, an antibody is an IgG4 antibody.

Modifications in the CH2 domain can affect the binding of FcRs to theFc. A number of amino acid modifications in the Fc region are known inthe art for selectively altering the affinity of the Fc for differentFc-gamma (Fcγ) receptors. In one embodiment, the Fc comprises one ormore modifications to promote selective binding of Fc-gamma receptors.

Exemplary mutations that alter the binding of FcRs to the Fc are listedbelow:

S298A/E333A/K334A, S298A/E333A/K334A/K326A (Lu Y, Vernes J M, Chiang N,et al. J Immunol Methods. 2011 Feb. 28; 365(1-2):132-41);

F243L/R292P/Y300L/V305I/P396L, F243L/R292P/Y300L/L235V/P396L(Stavenhagen J B, Gorlatov S, Tuaillon N, et al. Cancer Res. 2007 Sep.15; 67(18):8882-90; Nordstrom J L, Gorlatov S, Zhang W, et al. BreastCancer Res. 2011 Nov. 30; 13(6):R123);

F243L (Stewart R, Thom G, Levens M, et al. Protein Eng Des Sel. 2011September; 24(9):671-8.), S298A/E333A/K334A (Shields R L, Namenuk A K,Hong K, et al. J Biol Chem. 2001 Mar. 2; 276(9):6591-604);

S239D/I332E/A330L, S239D/I332E (Lazar G A, Dang W, Karki S, et al. ProcNatl Acad Sci USA. 2006 Mar. 14; 103(11):4005-10);

S239D/S267E, S267E/L328F (Chu S Y, Vostiar I, Karki S, et al. MolImnununol. 2008 September; 45(15):3926-33);

S239D/D265S/S298A/I332E, S239E/S298A/K326A/A327H,G237F/S298A/A330L/I332E, S239D/I332E/S298A,S239D/K326E/A330L/I332E/S298A, G236A/S239D/D270L/I332E,S239E/S267E/H268D, L234F/S267E/N325L, G237F/V266L/S267D and othermutations listed in WO2011/120134 and WO2011/120135, herein incorporatedby reference. Therapeutic Antibody Engineering (by William R. Strohl andLila M. Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1907568 37 9, October 2012) lists mutations on page 283.

In some embodiments an antibody described herein includes modificationsto improve its ability to mediate effector function. Such modificationsare known in the art and include afucosylation, or engineering of theaffinity of the Fc towards an activating receptor, mainly FCGR3a forADCC, and towards C1q for CDC. The Table B, below, summarizes variousdesigns reported in the literature for effector function engineering.

In certain embodiments, an antibody provided herein comprises an Fcregion with one or more amino acid substitutions which improve ADCC,such as a substitution at one or more of positions 298, 333, and 334 ofthe Fc region. In some embodiments, an antibody provided hereincomprises an Fc region with one or more amino acid substitutions atpositions 239, 332, and 330, as described in Lazar et al., Proc. Natl.Acad. Si. USA, 2006, 103:4005-4010, incorporated by reference in itsentirety.

In some embodiments, an antibody provided herein comprises one or morealterations that improves or diminishes C1q binding and/or CDC. See U.S.Pat. No. 6,194,551; WO 99/51642; and Idusogie et al., J. Immunol., 2000,164:4178-4184; each of which is incorporated by reference in itsentirety.

Thus, in one embodiment, an antibody described herein can include adimeric Fc that comprises one or more amino acid modifications as notedin Table B that confer improved effector function. In anotherembodiment, the antibody can be afucosylated to improve effectorfunction.

TABLE B CH2 domains and effector function engineering ReferenceMutations Effect Lu, 2011, Ferrara Afucosylated Increased ADCC 2011,Mizushima 2011 Lu, 2011 S298A/E333A/K334A Increased ADCC Lu, 2011S298A/E333A/K334A/ Increased ADCC K326A Stavenhagen, 2007F243L/R292P/Y300L/ Increased ADCC V305I/P396L Nordstrom, 2011F243L/R292P/Y300L/ Increased ADCC L235V/P396L Stewart, 2011 F243LIncreased ADCC Shields, 2001 S298A/E333A/K334A Increased ADCC Lazar,2006 S239D/I332E/A330L Increased ADCC Lazar, 2006 S239D/I332E IncreasedADCC Bowles, 2006 AME-D, not specified Increased ADCC mutations Heider,2011 37.1, mutations not Increased ADCC disclosed Moore, 2010S267E/H268F/S324T Increased CDC

Fc modifications reducing FcγR and/or complement binding and/or effectorfunction are known in the art. Recent publications describe strategiesthat have been used to engineer antibodies with reduced or silencedeffector activity (see Strohl, W R (2009), Curr Opin Biotech 20:685-691,and Strohl, W R and Strohl L M, “Antibody Fc engineering for optimalantibody performance” In Therapeutic Antibody Engineering, Cambridge:Woodhead Publishing (2012), pp 225-249). These strategies includereduction of effector function through modification of glycosylation,use of IgG2/IgG4 scaffolds, or the introduction of mutations in thehinge or CH2 regions of the Fc. For example, US Patent Publication No.2011/0212087 (Strohl). International Patent Publication No. WO2006/105338 (Xencor), US Patent Publication No. 2012/0225058 (Xencor),US Patent Publication No. 2012/0251531 (Genentech), and Strop et al((2012) J. Mol. Biol. 420; 204-219) describe specific modifications toreduce FcγR or complement binding to the Fc.

Specific, non-limiting examples of known amino acid modifications toreduce FcγR or complement binding to the Fc include those identified inthe following Table C:

TABLE C Modifications to reduce FcγR or complement binding to the FcCompany Mutations GSK N297A Ortho Biotech L234A/L235A Protein Designlabs IGG2 V234A/G237A Wellcome Labs IGG4 L235A/G237A/E318A GSK IGG4S228P/L236E Alexion IGG2/IGG4combo Merck IGG2 H268Q/V309L/A330S/A331SBristol-Myers C220S/C226S/C229S/P238S Seattle GeneticsC226S/C229S/E3233P/L235V/L235A Amgen E. coli production, non glycoMedimune L234F/L235E/P331S Trubion Hinge mutant, possibly C226S/P230S

Methods of producing antibodies with little or no fucose on the Fcglycosylation site (Asn 297 EU numbering) without altering the aminoacid sequence are well known in the art. The GlymaxX® technology(ProBioGen AG) is based on the introduction of a gene for an enzymewhich deflects the cellular pathway of fucose biosynthesis into cellsused for antibody production. This prevents the addition of the sugar“fucose” to the N-linked antibody carbohydrate part byantibody-producing cells. (von Horsten et al. (2010) Glycobiology. 2010December; 20 (12):1607-18.) Examples of cell lines capable of producingdefucosylated antibody include CHO-DG44 with stable overexpression ofthe bacterial oxidoreductase GDP-6-deoxy-D-lyxo-4-hexylose reductase(RMD) (see Henning von Horsten et al., Glycobiol 2010, 20:1607-1618) orLec13 CHO cells, which are deficient in protein fucosylation (see Ripkaet al., Arch. Biochem. Biophys., 1986, 249:533-545; U.S. Pat. Pub. No.2003/0157108; WO 2004/056312; each of which is incorporated by referencein its entirety), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene or FUT8 knockout CHO cells (seeYamane-Ohnuki et al., Biotech. Bioeng., 2004, 87; 614-622; Kanda et al.,Biotechnol. Bioeng., 2006, 94:680-688; and WO 2003/085107; each of whichis incorporated by reference in its entirety). Another approach toobtaining antibodies with lowered levels of fucosylation can be found inU.S. Pat. No. 8,409,572, which teaches selecting cell lines for antibodyproduction for their ability to yield lower levels of fucosylation onantibodies

Antibodies can be fully afucosylated (meaning they contain no detectablefucose) or they can be partially afucosylated, meaning that the isolatedantibody contains less than 95%, less than 85%, less than 75%, less than65%, less than 55%, less than 45%, less than 35%, less than 25%, lessthan 15% or less than 5% of the amount of fucose normally detected for asimilar antibody produced by a mammalian expression system.

In some aspects, an antibody provided herein comprises an IgG1 domainwith reduced fucose content at position Asn 297 compared to a naturallyoccurring IgG1 domain. Such Fc domains are known to have improved ADCC.See Shields et al., J. Biol. Chem., 2002, 277:26733-26740, incorporatedby reference in its entirety. In some aspects, such antibodies do notcomprise any fucose at position Asn 297. The amount of fucose may bedetermined using any suitable method, for example as described in WO2008/077546, incorporated by reference in its entirety.

In some embodiments, an antibody provided herein comprises a bisectedoligosaccharide, such as a biantennary oligosaccharide attached to theFc region of the antibody that is bisected by GlcNAc. Such antibodyvariants may have reduced fucosylation and/or improved ADCC function.Examples of such antibody variants are described, for example, in WO2003/011878; U.S. Pat. No. 6,602,684; and U.S. Pat. Pub. No.2005/0123546; each of which is incorporated by reference in itsentirety.

Other illustrative glycosylation variants which may be incorporated intothe antibodies provided herein are described, for example, in U.S. Pat.Pub. Nos. 2003/0157108, 2004/0093621, 2003/0157108, 2003/0115614,2002/0164328, 2004/0093621, 2004/0132140, 2004/0110704, 2004/0110282,2004/0109865; International Pat. Pub. Nos. 2000/61739, 2001/29246,2003/085119, 2003/084570, 2005/035586, 2005/035778; 2005/053742,2002/031140; Okazaki et al., J. Mol. Biol., 2004, 336:1239-1249; andYamane-Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; each of whichis incorporated by reference in its entirety.

In some embodiments, an antibody provided herein comprises an Fc regionwith at least one galactose residue in the oligosaccharide attached tothe Fc region. Such antibody variants may have improved CDC function.Examples of such antibody variants are described, for example, in WO1997/30087; WO 1998/58964; and WO 1999/22764; each of which hisincorporated by reference in its entirety.

Examples of cell lines capable of producing defucosylated antibodiesinclude CHO-DG44 with stable overexpression of the bacterialoxidoreductase GDP-6-deoxy-D-lyxo-4-hexylose reductase (RMD) (seeHenning von Horsten et al., Glycobiol 2010, 20:1607-1618) or Lec13 CHOcells, which are deficient in protein fucosylation (see Ripka et al.,Arch. Biochem. Biophys., 1986, 249:533-545; U.S. Pat. Pub. No.2003/0157108; WO 2004/056312; each of which is incorporated by referencein its entirety), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene or FUT8 knockout CHO cells (seeYamane-Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; Kanda et al.,Biotechnol. Bioeng., 2006, 94:680-688; and WO 2003/085107; each of whichis incorporated by reference in its entirety).

In some embodiments, an antibody has antibody-dependent cellularphagocytosis (ADCP) activity. ADCP can occur when antibodies bind toantigens on the surface of pathogenic or tumorigenic target-cells.Phagocytic cells bearing Fc receptors on their cell surface, includingmonocytes and macrophages, recognize and bind the Fc region ofantibodies bound to target-cells. Upon binding of the Fc receptor to theantibody-bound target cell, phagocytosis of the target cell can beinitiated. ADCP can be considered a form of ADCC.

In some embodiments, the antibodies are capable of forming an immunecomplex. For example, an immune complex can be a tumor cell covered byantibodies.

In some aspects, an anti-TREM2 antibody does not substantially bindmyeloid cells present outside of cancer tissue. In some aspects, ananti-TREM2 antibody does not substantially bind stimulatory myeloidcells present in cancer tissue.

In some embodiments the antibodies are monoclonal antibodies.

In some embodiments the antibodies are polyclonal antibodies.

In some embodiments the antibodies are produced by hybridomas. In otherembodiments, the antibodies are produced by recombinant cells engineeredto express the desired variable and constant domains.

In some embodiments the antibodies may be single chain antibodies orother antibody derivatives retaining the antigen specificity and thelower hinge region or a variant thereof.

In some embodiments the antibodies may be polyfunctional antibodies,recombinant antibodies, human antibodies, humanized antibodies,fragments or variants thereof. In particular embodiments, the antibodyfragment or a derivative thereof is selected from a Fab fragment, aFab′2 fragment, a CDR and ScFv.

In some embodiments, antibodies are specific for surface antigens, suchas TREM2 protein. In some embodiments, therapeutic antibodies arespecific for tumor antigens (e.g., molecules specifically expressed bytumor cells). In particular embodiments, the therapeutic antibodies mayhave human or non-human primate IgG1 or IgG3 Fc portions.

Binding

With regard to the binding of an antibody to a target molecule, theterms “bind.” “specific binding,” “specifically binds to.” “specificfor,” “selectively binds,” and “selective for” a particular antigen(e.g., a polypeptide target) or an epitope on a particular antigen meanbinding that is measurably different from a non-specific ornon-selective interaction (e.g., with a non-target molecule). Specificbinding can be measured, for example, by measuring binding to a targetmolecule and comparing it to binding to a non-target molecule. Specificbinding can also be determined by competition with a control moleculethat mimics the epitope recognized on the target molecule. In that case,specific binding is indicated if the binding of the antibody to thetarget molecule is competitively inhibited by the control molecule.

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen or epitope). Unlessindicated otherwise, as used herein, “affinity” refers to intrinsicbinding affinity, which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen or epitope). The affinity of amolecule X for its partner Y can be represented by the dissociationequilibrium constant (K_(D)). The kinetic components that contribute tothe dissociation equilibrium constant are described in more detailbelow. Affinity can be measured by common methods known in the art,including those described herein, such as surface plasmon resonance(SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g.,FORTEBIO®).

The term “k_(d)” (sec⁻¹), as used herein, refers to the dissociationrate constant of a particular antibody-antigen interaction. This valueis also referred to as the k_(off) value.

The term “k_(a)” (M⁻¹×sec⁻¹), as used herein, refers to the associationrate constant of a particular antibody-antigen interaction. This valueis also referred to as the k_(on) value.

The term “K_(D)” (M), as used herein, refers to the dissociationequilibrium constant of a particular antibody-antigen interaction.K_(D)=k_(d)/k_(a). In some embodiments, the affinity of an antibody isdescribed in terms of the K_(D) for an interaction between such antibodyand its antigen. For clarity, as known in the art, a smaller K_(D) valueindicates a higher affinity interaction, while a larger K_(D) valueindicates a lower affinity interaction.

The term “K_(A)” (M⁻¹), as used herein, refers to the associationequilibrium constant of a particular antibody-antigen interaction.K_(A)=k_(a)/k_(d).

When used herein in the context of two or more antibodies, the term“competes with” or “cross-competes with” indicates that the two or moreantibodies compete for binding to an antigen (e.g., TREM2). In oneexemplary assay, TREM2 is coated on a surface and contacted with a firstTREM2 antibody, after which a second TREM2 antibody is added. In anotherexemplary assay, a first TREM2 antibody is coated on a surface andcontacted with TREM2, and then a second TREM2 antibody is added. If thepresence of the first TREM2 antibody reduces binding of the second TREM2antibody, in either assay, then the antibodies compete with each other.The term “competes with” also includes combinations of antibodies whereone antibody reduces binding of another antibody, but where nocompetition is observed when the antibodies are added in the reverseorder. However, in some embodiments, the first and second antibodiesinhibit binding of each other, regardless of the order in which they areadded. In some embodiments, one antibody reduces binding of anotherantibody to its antigen by at least 25%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, or at least 95%. Askilled artisan can select the concentrations of the antibodies used inthe competition assays based on the affinities of the antibodies forTREM2 and the valency of the antibodies. The assays described in thisdefinition are illustrative, and a skilled artisan can utilize anysuitable assay to determine if antibodies compete with each other.Suitable assays are described, for example, in Cox et al., “lmmunoassayMethods,” in Assay Guidance Manual [Internet], Updated Dec. 24, 2014(www.ncbi.nlm.nih.gov/books/NBK92434/; accessed Sep. 29, 2015); Silmanet al., Cytometry, 2001, 44:30-37; and Finco et al., J. Pharm. BiomedAnal., 2011, 54:351-358; each of which is incorporated by reference inits entirety.

In some embodiments, an antibody provided herein binds human TREM2 witha K_(D) of less than or equal to about 0.001, 0.01, 0.02, 0.03, 0.04,0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.95, 2, 3, 4, 5,6, 7, 8, 9, or 10×10⁻⁹ M, as measured by Biacore assay. In someembodiments, the KC of the antibody provided herein is between about0.001-0.01, 0.01-0.1, 0.01-0.05, 0.05-0.1, 0.1-0.5, 0.5-1, 0.25-0.75,0.25-0.5, 0.5-0.75, 0.75-1, 0.75-2, 1.1-1.2, 1.2-1.3, 1.3-1.4, 1.4-1.5,1.5-1.6, 1.6-1.7, 1.7-1.8, 1.8-1.9, 1.9-2, 1-2, 1-5, 2-7, 3-8, 3-5, 4-6,5-7, 6-8, 7-9, 7-10, or 5-10×10⁻⁹ M, as measured by Biacore assay.

In some embodiments, the antibody provided herein binds human TREM2 witha K_(D) of less than or equal to about 2, 1.98, 1.95, 1.9, 1.85, 1.8,1.75, 1.7, 1.65, 1.6, 1.55, 1.50, 1.45, or 1.4×10⁻⁹ M, or less, asmeasured by Biacore assay. In some embodiments, the antibody providedherein binds human TREM2 with a Ku between 1.9-1.8, 1.8-1.7, 1.7-1.6,1.6-1.5, or 1.9-1.5×10⁻⁹ M as measured by Biacore assay. In someembodiments, the antibody provided herein binds human TREM2 with a k_(d)of less than or equal to about 10, 9.56, 9.5, 9.0, 8.88, 8.84, 8.5, 8,7.5, 7.32, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1×10⁻⁴(l/s), or less, as measured by Biacore assay. In some embodiments, theantibody provided herein binds human TREM2 with a K_(d) between 7-10,7-8, 8-9, 9-10, 7-7.5, 7.5-8, 8.-8.5, 8.5-9, 9-9,5, or 9.5-10×10⁻⁴ (l/s)as measured by Biacore assay. In some embodiments, the antibody providedherein binds human TREM2 with a K of greater than or equal to about 4,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 45, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6, 7, 8, 9, or 10×10⁵ (l/Ms), or more, asmeasured by Biacore assay. In some embodiments, the antibody providedherein binds human TREM2 with a K, between 4-7, 4-4.5, 4.5-5, 5-5.5,5.5-6, 6-6.5, or 6.5-7, 7-8, 8-9, or 9-10×10⁵(l/Ms) as measured byBiacore assay.

In some embodiments, the antibody provided herein binds human TREM2 withan EC50 of less than or equal to 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3,1.2, 1.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 nM as measuredby measured by flow cytometry. In some embodiments, the antibody bindshuman TREM2 with an EC50 between 0.6-1.4 nM as measured by measured byflow cytometry. In some embodiments, the antibody binds human TREM2 withan EC50 of about 0.5, 0.6, 0.9, 1.1, 1.2, 1.3, 1.4, or 1.5 nM asmeasured by measured by flow cytometry.

In some embodiments, the antibody provided herein binds mouse TREM2 withan EC50 of less than or equal to 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3,1.2, 1.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 nM as measuredby measured by flow cytometry. In some embodiments, the antibody bindsmouse TREM2 with an EC50 between 0.6-1.4 nM as measured by measured byflow cytometry. In some embodiments, the antibody binds mouse TREM2 withan EC50 of about 0.5, 0.6, 0.9, 1.1, 1.2, 1.3, 1.4, or 1.5 nM asmeasured by measured by flow cytometry.

In some embodiments, the antibody provided herein does not bind humanTREM2 with an EC50 great than or equal to 20 nM or more as measured bymeasured by flow cytometry. In some embodiments, the antibody providedherein does not bind mouse TREM2 with an EC50 great than or equal to 3nM or more as measured by measured by flow cytometry.

To screen for antibodies which bind to an epitope on a target antigenbound by an antibody of interest (e.g., TREM2), a routine cross-blockingassay such as that described in Antibodies, A Laboratory Manual, ColdSpring Harbor Laboratory, Ed Harlow and David Lane (1988), can beperformed. Alternatively, or additionally, epitope mapping can beperformed by methods known in the art.

Competition between antibodies can be determined by an assay in which anantibody under test inhibits or blocks specific binding of a referenceantibody to a common antigen (see, e.g., Junghans et al., Cancer Res.50:1495, 1990; Fendly et al. Cancer Research 50: 1550-1558; U.S. Pat.No. 6,949,245). A test antibody competes with a reference antibody if anexcess of a test antibody (e.g., at least 2×, 5×, 10×, 20×, or 100×)inhibits or blocks binding of the reference antibody by, e.g., at least50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as measured in acompetitive binding assay. Antibodies identified by competition assay(competing antibody) include antibodies binding to the same epitope asthe reference antibody and antibodies binding to an adjacent epitopesufficiently proximal to the epitope bound by the reference antibody forsteric hindrance to occur. For example, a second, competing antibody canbe identified that competes for binding to TREM2 with a first antibodydescribed herein. In certain instances, the second antibody can block orinhibit binding of the first antibody by, e.g., at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, or 99% as measured in a competitive bindingassay. In certain instances, the second antibody can displace the firstantibody by greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

Function

In some embodiments, the antibody has antibody-dependent cellularcytotoxicity (ADCC) activity. ADCC can occur when antibodies bind toantigens on the surface of pathogenic or tumorigenic target-cells.Effector cells bearing Fc gamma receptors (FcγR or FCGR) on their cellsurface, including cytotoxic T-cells, natural killer (NK) cells,macrophages, neutrophils, eosinophils, dendritic cells, or monocytes,recognize and bind the Fc region of antibodies bound to thetarget-cells. Such binding can trigger the activation of intracellularsignaling pathways leading to cell death. In particular embodiments, theantibody's immunoglobulin Fc region subtypes (isotypes) include humanIgG1 and IgG3. As used herein, ADCC refers to a cell-mediated reactionin which nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognizebound antibody on a target cell and subsequently cause lysis of thetarget cell. The primary cells for mediating ADCC, NK cells, expressFcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcRexpression on hematopoietic cells in summarized is Table 3 on page 464of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCCactivity of a molecule of interest, an in vitro ADCC assay, such as thatdescribed in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed.Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in an animal model such as that disclosed inClynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998).

In some embodiments, the antibody has complement-dependent cytotoxicity(CDC) activity. Antibody-induced CDC is mediated through the proteins ofthe classical complement cascade and is triggered by binding of thecomplement protein C1q to the antibody. Antibody Fc region binding toC1q can induce activation of the complement cascade. In particularembodiments, the antibody's immunoglobulin Fc region subtypes (isotypes)include human IgG1 and IgG3. As used herein, CDC refers to the abilityof a molecule to lyse a target in the presence of complement. Thecomplement activation pathway is initiated by the binding of the firstcomponent of the complement system (C1q) to a molecule (e.g. polypeptide(e.g., an antibody)) complexed with a cognate antigen. To assesscomplement activation, a CDC assay, e.g. as described in Gazzano-Santoroet al., J. Immunol. Methods 202:163 (1996), may be performed.

In some embodiments, an antibody is an agonistic antibody. An agonisticantibody can induce (e.g., increase) one or more activities or functionsof NSMs after the antibody binds a TREM2 protein expressed on the cell.The agonistic antibody may bind to and activate NSMs, causing changes inproliferation of the cell or modifying antigen presentationcapabilities. The agonistic antibody may bind to and activate NSMs,triggering intracellular signaling pathways that lead to modified cellgrowth or apoptosis.

In some embodiments, an antibody is an antagonistic antibody. Anantagonistic antibody can block (e.g. decrease) one or more activitiesor functions of NSMs after the antibody binds a TREM2 protein expressedon the cell. For example, the antagonist antibody may bind to and blockligand binding to one or more NSM proteins, preventing differentiationand proliferation of the cell or modifying antigen presentationcapabilities. The antagonist antibody may bind to and prevent activationof a TREM2 protein by its ligand, modifying intracellular signalingpathways that contribute to cell growth and survival.

In some embodiments an antibody is a depleting antibody. A depletingantibody is one that would kill a non-stimulatory myeloid cell uponcontact through the antibody's interaction with other immune cells ofmolecules. For example, antibodies, when bound to cells bearing TREM2proteins, could engage complement proteins and inducecomplement-dependent cell lysis. Antibodies, when bound to cells bearingTREM2 proteins, could also trigger neighboring cells bearing Fcreceptors to kill them by antibody-dependent cellular cytotoxicity(ADCC).

In some embodiments, an antibody is a neutralizing antibody, and theantibody neutralizes one or more biological activities of NSMs. In someembodiments, TREM2 protein is expressed on the surface ofnon-stimulatory myeloid cells and the antibody recognizes theextracellular domain of TREM2 protein.

In some embodiments an antibody is selective for NSMs (preferentiallybinds to TREM2). In certain embodiments, an antibody that selectivelybinds to NSMs has a dissociation constant (Kd) of range of 0.0001 nM to1 μM. In certain embodiments, an antibody specifically binds to anepitope on a TREM2 protein that is conserved among the protein fromdifferent species. In another embodiment, selective binding includes,but does not require, exclusive binding.

In one embodiment an anti-TREM2 antibody bound to its target isresponsible for causing the in vivo depletion of non-stimulatory myeloidcells to which it is bound. In some embodiments, effector proteinsinduced by clustered antibodies can trigger a variety of responses,including release of inflammatory cytokines, regulation of antigenproduction, endocytosis, or cell killing. In one embodiment the antibodyis capable of recruiting and activating complement or mediatingantibody-dependent cellular cytotoxicity (ADCC) in vivo, or mediatingphagocytosis by binding Fc receptors in vivo. The antibody may alsodeplete non-stimulatory myeloid cells by inducing apoptosis or necrosisof the non-stimulatory myeloid cell upon binding.

In some embodiments the disabling of non-stimulatory myeloid cells is invitro and is achieved: a) by killing of the non-stimulatory myeloidcells; b) magnetic bead depletion of the non-stimulatory myeloid cells;or c) Fluorescence-activated cell sorting (FACS) sorting of thenon-stimulatory myeloid cells.

In some embodiments, an antibody is bound to, or conjugated to aneffector molecule. In particular embodiments, an antibody is conjugatedto at least one therapeutic agent selected from the group consisting ofa radionuclide, a cytotoxin, a chemotherapeutic agent, a drug, apro-drug, a toxin, an enzyme, an immunomodulator, an anti-angiogenicagent, a pro-apoptotic agent, a cytokine, a hormone, an oligonucleotide,an antisense molecule, a siRNA, a second antibody and a second antibodyfragment.

In certain embodiments an antibody is conjugated to a drug, e.g., atoxin, a chemotherapeutic agent, an immune modulator, or a radioisotope.Several methods of preparing ADCs (antibody drug conjugates) are knownin the art and are described in U.S. Pat. No. 8,624,003 (pot method),U.S. Pat. No. 8,163,888 (one-step), and U.S. Pat. No. 5,208,020(two-step method), for example. An antibody or antigen-binding fragmentthereof can be conjugated to at least one agent including aradionuclide, a cytotoxin, a chemotherapeutic agent, a drug, a pro-drug,a toxin, an enzyme, an immunomodulator, an anti-angiogenic agent, apro-apoptotic agent, a cytokine, a hormone, an oligonucleotide, anantisense molecule, a siRNA, a second antibody, and a second antibodyfragment that is antigen binding.

Non-stimulatory Myeloid Cells (NSMs)

Provided herein are methods and compositions for disabling and/ordetecting non-stimulatory myeloid cells (NSMs) comprising the use of ananti-TREM2 antibody. Also provided herein are methods and compositionsfor targeting and/or detecting non-stimulatory myeloid cells expressinga NSM protein.

Also provided herein are methods and compositions for disabling and/ordetecting non-stimulatory myeloid cells comprising the use of antibodydirected at a non-human homolog of human NSM protein, in that non-humanindividual.

As used herein, non-stimulatory myeloid cells are myeloid cells that arenot sufficiently effective at stimulating an immune response (e.g. notas effective at stimulating an anti-tumor response in a tumormicroenvironment compared to stimulatory myeloid cells). In someembodiments, non-stimulatory myeloid cells are not as effective atpresenting an antigen (e.g. a tumor antigen) to T-cells or not aseffective at stimulating tumor specific T-cell responses as compared toa stimulatory myeloid cell. In some embodiments, non-stimulatory myeloidcells can display a decreased ability to uptake, process, and/or presenttumor-associated antigens to a T cell as compared to a stimulatorymyeloid cell. Non-stimulatory myeloid cells may contain a reducedability or no ability to re-prime cytotoxic T lymphocytes or in somecases cannot stimulate effective tumor-cell killing. Non-stimulatorymyeloid cells may display lower expression of gene and cell-surfacemarkers involved in antigen processing, antigen presentation and/orantigen co-stimulation including, without limitation. CD80, CD86, MHCI,and MHCII compared to stimulatory myeloid cells.

Non-stimulatory myeloid cells, when compared to stimulatory myeloidcells, may display the lower expression of genes associated withcross-presentation, co-stimulation, and/or stimulatory cytokines,including, without limitation, any one or more of TAP1, TAP2, PSMB8,PSMB9, TAPBP, PSME2, CD24a, CD274, BTLA, CD40, CD244, ICOSL, ICAM1,TIM3, PDL2, RANK, FLT3, CSF2RB, CSF2RB2, CSF2RA, IL12b, XCR1, CCR7,CCR2, CCL22, CXCL9, and CCL5, and increased expression ofanti-inflammatory cytokine IL-10. In some embodiments, non-stimulatorymyeloid cells are dependent on the transcription factor IRF4 and thecytokines GM-CSF or CSF-1 for differentiation and survival. In someembodiments, non-stimulatory myeloid cells can contribute to tumoralangiogenesis by secreting vascular endothelial growth factor (VEGF) andnitric oxide synthase (NOS) and support tumor growth by secretingepidermal growth factor (EGF).

In some embodiments, non-stimulatory myeloid cells are tumor-associatedmacrophages (TAM), neutrophils, monocytes, or dendritic cells (DC). Insome embodiments, the non-stimulatory myeloid cell is not a dendriticcell (DC). In some embodiments, non-stimulatory myeloid cells areneutrophils.

In some embodiments, non-stimulatory myeloid cells are tumor-associatedmacrophages (TAMs). TAMs are macrophages present near or withincancerous tumors, and are derived from circulating monocytes or residenttissue macrophages.

In some embodiments the non-stimulatory myeloid cells and thestimulatory myeloid cells are distinguished on the basis of the markersthey express, or the markers they selectively express. The expression ofa cell surface markers can be described as ‘+’ or ‘positive’. Theabsence of a cell surface marker can be described as ‘−’ or ‘negative’.The expression of a cell surface marker can be further described as‘high’ (cells expressing high levels of the makers) or ‘low’ (cellsexpressing low levels of the markers), which indicates the relativeexpression of each marker on the cell surface. The level of markers maybe determined by various methods known in the art, e.g. immuno-stainingand FACS analysis, or gel electrophoresis and Western blotting.

In some embodiments, non-stimulatory myeloid cells are dendritic cells(DCs). In some embodiments, dendritic cells can be distinguished by aspikey or dendritic morphology. In one embodiment, the non-stimulatorydendritic cell is at least CD45+, HLA-DR+, CD14−, CD11c+, and BDCA+(also referred to as DC1 cells). In one embodiment, the non-stimulatorydendritic cell is not CD45+, HLA-DR+, CD14−, CD11c+, and BDCA3+ (alsoreferred to as DC2 cells). In one embodiment, a dendritic cell that isCD45+, HLA-DR+, CD14−, CD11c+, and BDCA3+ is a stimulatory-myeloid cell.

In some embodiments, non-stimulatory myeloid cells are tumor associatedmacrophages. In some embodiments, for example in humans, thenon-stimulatory tumor associated macrophages are at least CD45+,HLA-DR+, CD14+. In some embodiments the non-stimulatory tumor associatedmacrophages are at least CD45⁺, HLA-DR⁺, CD14⁺, CD11b⁺. In someembodiments the non-stimulatory tumor associated macrophages are atleast CD45⁺, HLA-DR⁺, CD14⁺, CD11c⁺. In some embodiments thenon-stimulatory tumor associated macrophages are at least CD45⁺,HLA-DR⁺, CD14⁺, BDCA3⁻. In some embodiments the non-stimulatory tumorassociated macrophages are at least CD45⁺, HLA-DR⁺, CD14⁺, BDCA3⁻,CD11b⁺. In some embodiments the non-stimulatory tumor associatedmacrophages are at least CD45⁺, HLA-DR⁺, CD14⁺, BDCA3⁻, CD11c⁺. In someembodiments the non-stimulatory tumor associated macrophages are atleast CD45⁺, HLA-DR⁺, CD14⁺, CD11b⁺, and CD11c⁺. In some embodiments thenon-stimulatory tumor associated macrophages are at least CD45⁺,HLA-DR⁺, CD14⁺, BDCA3⁻, CD11b⁺, and CD11c⁺.

In some embodiments the methods and compositions of the presentinvention are useful for targeting TAMs and DCs in other mammals, forexample in mice. In such embodiments, mice TAMs and DCs are contactedwith a TREM2 antibody. In one embodiment, for example in mice, thetumor-associated macrophage is at least CD45+, HLA-DR+, CD14+,CD11b^(high), and CD11c^(low) (also referred to as TAM1). In oneembodiment, for example in mice, tumor-associated macrophages are atleast CD45+, HLA-DR+, CD14+, CD11b^(low), and CD11c^(high) (alsoreferred to as TAM2). The term “CD11b^(high) macrophages”, as usedherein, relates to macrophages expressing high levels of CD11b. The term“CD11b^(low) macrophages,” as used herein, relates to macrophages thatexpress on their surface a level of CD11b that is substantially lowerthan that of CD11b^(high) macrophages. The term “CD11c^(high)”, as usedherein, relates to macrophages expressing high levels of CD11c. The term“CD11c^(low) macrophages”, as used herein, relates to macrophages thatexpress on their surface a level of CD11c that is substantially lowerthan that of Cd11c^(high) macrophages.

In some embodiments, the non-stimulatory myeloid cells of the inventioninclude one or more of TAM and DC cells.

In some embodiments, for example in mice, the non-stimulatory myeloidcells of the invention include one or more of TAM1, TAM2, and DC1 cells.In such embodiments the non-stimulatory myeloid cells of the inventionare contacted with a TREM2 antibody.

In some embodiments, the non-stimulatory myeloid cells are myeloid cellsare intratumoral.

In some embodiments, the non-stimulatory myeloid cells are localizedwithin the margins of the tumoral lesions or in the transformed tumorducts, where they come into contact with cognate T-cells. In oneembodiment, the localization of the non-stimulatory myeloid cell ismodified, so that the cells are no longer localized at the tumor marginor are no longer in contact with T-cells.

In some embodiments, the non-stimulatory myeloid cells are in apopulation of immune cells comprising stimulatory myeloid cells andnon-stimulatory myeloid cells. In some embodiments, the non-stimulatorymyeloid cells are in a population of immune cells comprising onlynon-stimulatory myeloid cells. The populations of immune cells of thepresent invention may be pure, homogenous, heterogeneous, derived from avariety of sources (e.g. diseased tissue, tumor tissue, healthy tissue,cell banks), maintained in primary cell cultures, maintained inimmortalized cultures, and/or maintained in ex vivo cultures.

In some embodiments, the non-stimulatory myeloid cells aretumor-associated macrophages.

In some embodiments, the non-stimulatory myeloid cells are dendriticcells.

In some embodiments, the non-stimulatory myeloid cells are CD45⁺,HLA-DR⁺, CD14⁻, CD11c⁺, and BDCA1′. In some embodiments, thenon-stimulatory myeloid cells comprise cells that are CD45⁺, HLA-DR⁺,CD14⁻, CD11c⁺, and BDCA1⁺. In some embodiments, the non-stimulatorymyeloid cells consist of cells that are CD45⁺, HLA-DR⁺, CD14⁻, CD11c⁺,and BDCA1⁺. In some embodiments, the non-stimulatory myeloid cellsconsist essentially of cells that are CD45⁺, HLA-DR⁺, CD14⁻, CD11c⁺, andBDCA1⁺.

In some embodiments, the non-stimulatory myeloid cells are CD45⁺,HLA-DR⁺, CD14⁺, BDCA3⁻. In some embodiments, the non-stimulatory myeloidcells comprise cells that are CD45⁺, HLA-DR⁺, CD14⁺, BDCA3⁻. In someembodiments, the non-stimulatory myeloid cells consist of cells that areCD45⁺, HLA-DR⁺, CD14⁺, BDCA3⁻. In some embodiments, the non-stimulatorymyeloid cells consist essentially of cells that are CD45⁺, HLA-DR⁺,CD14⁺, BDCA3⁻.

In some embodiments, the non-stimulatory myeloid cells are CD45⁺,HLA-DR⁺, CD14⁺, CD11b⁺. In some embodiments, the non-stimulatory myeloidcells comprise cells that are CD45⁺, HLA-DR⁺, CD14⁺, CD11b⁺. In someembodiments, the non-stimulatory myeloid cells consist of cells that areCD45⁺, HLA-DR⁺, CD14⁺, CD11b⁺. In some embodiments, the non-stimulatorymyeloid cells consist essentially of cells that are CD45⁺, HLA-DR⁺,CD14⁺, CD11b⁺.

In some embodiments, the non-stimulatory myeloid cells are CD45⁺,HLA-DR⁺, CD14⁺, CD11⁺. In some embodiments, the non-stimulatory myeloidcells comprise cells that are CD45⁺, HLA-DR⁺, CD14⁺, CD11c⁺. In someembodiments, the non-stimulatory myeloid cells consist of cells that areCD45⁺, HLA-DR⁺, CD14⁺, CD11c⁺. In some embodiments, the non-stimulatorymyeloid cells consist essentially of cells that are CD45⁺, HLA-DR⁺,CD14⁺, CD11c⁺.

In some embodiments, the non-stimulatory myeloid cells are CD45⁺,HLA-DR⁺, CD14⁺, BDCA3⁻, and CD11c⁺. In some embodiments, thenon-stimulatory myeloid cells comprise cells that are CD45⁺, HLA-DR⁺,CD14⁺, BDCA3⁻, and CD11c⁺. In some embodiments, the non-stimulatorymyeloid cells consist of cells that are CD45⁺, HLA-DR⁺, CD14⁺, BDCA3⁻,and CD11c⁺. In some embodiments, the non-stimulatory myeloid cellsconsist essentially of cells that are CD45⁺, HLA-DR⁺, CD14⁺, BDCA3⁻, andCD11c⁺.

In some embodiments, the non-stimulatory myeloid cells are CD45⁺,HLA-DR⁺, CD14⁺, BDCA3⁻, CD11b⁺. In some embodiments, the non-stimulatorymyeloid cells comprise cells that are CD45⁺, HLA-DR⁺, CD14⁺, BDCA3⁻,CD11b⁺. In some embodiments, the non-stimulatory myeloid cells consistof cells that are CD45⁺, HLA-DR⁺, CD14⁺, BDCA3⁻, CD11b⁺. In someembodiments, the non-stimulatory myeloid cells consist essentially ofcells that are CD45⁻, HLA-DR⁻, CD14⁺, BDCA3⁻, CD11b⁺.

In some embodiments, the non-stimulatory myeloid cells are CD45⁺,HLA-DR⁺, CD14⁺, CD11b⁺, and CD11c⁺. In some embodiments, thenon-stimulatory myeloid cells comprise cells that are CD45⁺, HLA-DR⁺,CD14⁺, CD11b⁺, and CD11c⁺. In some embodiments, the non-stimulatorymyeloid cells consist of cells that are CD45⁺, HLA-DR⁺, CD14⁺, CD11b⁺,and CD11c⁺. In some embodiments, the non-stimulatory myeloid cellsconsist essentially of cells that are CD45⁺, HLA-DR⁺, CD14⁺, CD11b⁺, andCD11c⁺.

In some embodiments, the non-stimulatory myeloid cells are CD45⁺,HLA-DR⁺, CD14⁺, BDCA3⁻, CD11b⁺, and CD11c⁺. In some embodiments, thenon-stimulatory myeloid cells comprise cells that are CD45⁺, HLA-DR⁺,CD14⁺, BDCA3⁻, CD11b⁺, and CD11c⁺. In some embodiments, thenon-stimulatory myeloid cells consist of cells that are CD45⁺, HLA-DR⁺,CD14⁺, BDCA3⁻, CD11b⁺, and CD11c⁺. In some embodiments, thenon-stimulatory myeloid cells consist essentially of cells that areCD45⁺, HLA-DR⁺, CD14⁺, BDCA3⁻, CD11b⁺, and CD11c⁺.

In some embodiments, the non-stimulatory myeloid cells are not CD45⁺,HLA-DR⁺, CD14⁻, CD11c⁺, and BDCA3⁺. In some embodiments, thenon-stimulatory myeloid cells comprise cells that are not CD45⁺,HLA-DR⁺, CD14⁻, CD11c⁺, and BDCA3⁺.

In some embodiments, for example in mice, the non-stimulatory myeloidcells are CD45⁺, HLA-DR⁺, CD14⁺, CD11b^(high), and CD11c^(low). In someembodiments, for example in mice, the non-stimulatory myeloid cellscomprise cells that are CD45⁺, HLA-DR⁺, CD14⁺, CD11b^(high), andCD11c^(low). In some embodiments, for example in mice, thenon-stimulatory myeloid cells consist of cells that are CD45⁺, HLA-DR⁺,CD14⁺, CD11b^(high), and CD11c^(low). In some embodiments, for examplein mice, the non-stimulatory myeloid cells consist essentially of cellsthat are CD45⁺, HLA-DR⁺, CD14⁺, CD11b^(high), and CD11c^(low). In suchembodiments the non-stimulatory mice myeloid cells are contacted with aTREM2 antibody.

In some embodiments, for example in mice, the non-stimulatory myeloidcells are CD45⁺, HLA-DR⁺, CD14⁺, CD11b^(high), and CD11c^(low). In someembodiments, for example in mice, the non-stimulatory myeloid cellscomprise cells that are CD45⁺, HLA-DR⁺, CD14⁺, CD11b^(high), andCD11c^(low). In some embodiments, for example in mice, thenon-stimulatory myeloid cells consist of cells that are CD45⁺, HLA-DR⁺,CD14⁺, CD11b^(high), and CD11c^(high). In some embodiments, for examplein mice, the non-stimulatory myeloid cells consist essentially of cellsthat are CD45⁺, HLA-DR⁺, CD14⁺, CD11b^(low), and CD11c^(high). In suchembodiments the non-stimulatory mice myeloid cells are contacted with aTREM2 antibody.

In some embodiments, the non-stimulatory myeloid cells are in a cancertissue.

In some embodiments, the population of immune cells is in a cancertissue.

In some embodiments, the non-stimulatory cells and stimulatory myeloidcells are in a cancer tissue.

In some embodiments, the biological sample comprises a population ofimmune cells comprising non-stimulatory myeloid cells and stimulatorymyeloid cells.

NSM cells can refer collectively to DC1, TAM1, and TAM2 cells present intumor tissues and which may be distinguished from other cell types bytheir expression of NSM cell markers. For example, genes and associatedproteins which are expressed or translated in greater abundance in NSMcells than SDC's may act as NSM markers. An exemplary NSM marker isCD11b. Additional exemplary NSM markers are listed in Table A. NSM cellscan express TREM2, MS4A7, C5AR1, LYVE1, ABCC3, LILRB4, MRC1/CD206,SIGLEC1, STAB1, TMEM37, MERTK, and TMEM119 on their cell surface. Insome aspects, NSM cells do not express at least one of KIT, CCR7, BATF3,FLT3, ZBTB46, IRF8, BTLA, MYCL1, CLEC9A, BDCA3, and XCR1.

In one embodiment, NSM cells express one or more of the NSM marker geneslisted in Table A. In another embodiment. NSM cells express 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more of the NSMmarkers listed in Table D. In another embodiment, NSM cells express mostor all of the NSM markers listed in Table D. In another embodiment, NSMcells are identified as expressing MRC1, MS4A7, C1QC, APOE, C1QB, C1QA,and C5AR1.

TABLE D SDC Markers NSM Markers KIT C5AR1 CCR7 LYVE1 BATF3 ABCC3 FLT3MRC1 ZBTB46 SIGLEC1 IRF8 STAB1 BTLA C1QB MYCL1 C1QA CLEC9A TMEM37 BDCA3MERTK XCR1 C1QC TMEM119 MS4A7 APOE CYP4F18 TREM2 TLR7 LILRB4

Stimulatory Myeloid Cells

As used herein, stimulatory myeloid cells (also called SDCs in certainaspects) are myeloid cells that are effective at stimulating an immuneresponse (e.g. more effective at stimulating an anti-tumor response in atumor microenvironment compared to non-stimulatory myeloid cells). Insome embodiments, stimulatory myeloid cells are effective at presentingan antigen (e.g. a tumor antigen) to T-cells or are effective atstimulating tumor specific T-cell responses as compared to anon-stimulatory myeloid cell. In some embodiments, stimulatory myeloidcells can display an increased ability to uptake, process, and/orpresent tumor-associated antigens to a T cell as compared to anon-stimulatory myeloid cell. Stimulatory myeloid cells can have anincreased ability to re-prime cytotoxic T lymphocytes or in some casesstimulate effective tumor-cell killing relative to non-stimulatorymyeloid cells. Stimulatory myeloid cells may display higher expressionof gene and cell-surface markers involved in antigen processing, antigenpresentation and/or antigen co-stimulation including, withoutlimitation, CD80, CD86, MHCI, and MHCII compared to non-stimulatorymyeloid cells.

Exemplary stimulatory myeloid cell markers are listed in Table A. Forexample, in human SDC's, the expression of Xcr1, Clec9a, and BDCA3(CD141) are markers of SDC identity. It will be noted that in mice,CD103 can also be used as a strong marker of SDC identity, although itis not expressed in human SDC's.

In one embodiment, SDC's are tumor infiltrating myeloid cells havingdendritic cell identity and which also express one or more of the SDCmarkers listed in Table A. In another embodiment, SDC's are tumorinfiltrating myeloid cells having dendritic cell identity and which alsoexpress two, three, four, five, six, seven, eight, nine or all of theSDC markers listed in Table A. In another embodiment. SDC's areidentified as tumor infiltrating myeloid dendritic cells expressingBDCA3, KIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, XCR1 andCLEC9A. SDC's cells can express at least one of KIT, CCR7, BATF3, FLT3,ZBTB46, IRF8, BTLA, MYCL1, CLEC9A, BDCA3, and XCR1. In some embodiments,SDC's do not substantially express TREM2, MS4A7, C5AR1, LYVE1, ABCC3,LILRB4, MRC1/CD206, SIGLEC1, STAB1, TMEM37, MERTK, and/or TMEM119 ontheir cell surface. In some embodiments, SDC's do not substantiallyexpress C5AR1, LYVE1, ABCC3, MRC1, SIGLEC1, STAB1, C1QB, C1QA, TMEM37,MERTK, C1QC, TMEM119, MS4A7, APOE, CYP4F18, TREM2, TLR7, and/or LILRB4.Flow cytometry and PCR, among other art recognized assays, can be usedto assess expression of a marker disclosed herein.

Stimulatory myeloid cells can be CD45⁺, HLA-DR⁺, CD14⁻, CD11c⁺, andBDCA3⁺. Stimulatory myeloid cells can be CD45⁺, HLA-DR⁺, and BDCA3⁺.Stimulatory myeloid cells can be CD45⁺, HLA-DR⁺, CD14⁻, and BDCA3′.Stimulatory myeloid cells can be CD45⁺, HLA-DR⁺, CD11c⁺, and BDCA3⁺.

Proteins, Nucleotides, and Homologs

Provided herein are methods and compositions for disabling and/ordetecting non-stimulatory human myeloid cells that express NSM proteins.In some embodiments, the invention is directed to disabling and/ordetecting non-stimulatory myeloid cells from non-human mammalian cellsthat express a NSM protein homolog. For example, NSM proteins in themouse can express a comparable restricted pattern of expression as itshuman homolog. Thus in one embodiment, provided herein are methods andcompositions for disabling and/or detecting non-stimulatory mousemyeloid cells that express an NSM protein. Also provided herein aresimilar methods and compositions for disabling and/or detectingnon-stimulatory cells from any individual that expresses a homolog of aNSM protein, with a similar expression pattern, which cells exhibit acomparable pattern of expression as that of the NSM protein.

NSM proteins or nucleotides can include at least one or more of C5AR1,LYVE1, ABCC3, MRC1, SIGLEC1, STAB1, C1QB, C1QA, TMEM37, MERTK, C1QC,TMEM19, MS4A7, APOE, CYP4F18, TREM2, TLR7, and LILRB4, and homologsthereof. SDC proteins or nucleotides can include at least one or more ofKIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, CLEC9A, BDCA3, andXCR1, and homologs thereof. Cell surface NSM proteins can include atleast one or more of TREM2, MS4A7, C5AR1, LYVE1, ABCC3, LILRB4,MRC1/CD206, SIGLEC1, STAB1, TMEM37, MERTK, and TMEM19. Cell surface NSMproteins can be targeted by one or more anti-TREM2 antibodies, alone orin combination. Generally NSMs are positive for NSM proteins ornucleotides and negative for SDC proteins or nucleotides; converselySDCs are generally positive for SDC proteins or nucleotides and negativefor NSM proteins or nucleotides.

The antibodies described herein comprise at least one polypeptide,though they typically comprise a dimer of a HC/LC, i.e., fourpolypeptides. Also described are polynucleotides encoding thepolypeptides described herein. The antibodies are typically isolated.

As used herein, “isolated” means an agent (e.g., a polypeptide orpolynucleotide) that has been identified and separated and/or recoveredfrom a component of its natural cell culture environment. Contaminantcomponents of its natural environment are materials that would interferewith diagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.Isolated also refers to an agent that has been synthetically produced,e.g., via human intervention.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally encoded amino acid. As used herein, the terms encompassamino acid chains of any length, including full length proteins, whereinthe amino acid residues are linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, praline, serine, threonine, tryptophan,tyrosine, and valine) and pyrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., a carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Reference to an amino acidincludes, for example, naturally occurring proteogenic L-amino acids;D-amino acids, chemically modified amino acids such as amino acidvariants and derivatives; naturally occurring non-proteogenic aminoacids such as β-alanine, ornithine, etc.; and chemically synthesizedcompounds having properties known in the art to be characteristic ofamino acids. Examples of non-naturally occurring amino acids include,but are not limited to, α-methyl amino acids (e.g. α-methyl alanine),D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine,β-hydroxy-histidine, homohistidine), amino acids having an extramethylene in the side chain (“homo” amino acids), and amino acids inwhich a carboxylic acid functional group in the side chain is replacedwith a sulfonic acid group (e.g., cysteic acid). The incorporation ofnon-natural amino acids, including synthetic non-native amino acids,substituted amino acids, or one or more D-amino acids into the proteinsof the present invention may be advantageous in a number of differentways. D-amino acid-containing peptides, etc., exhibit increasedstability in vitro or in vivo compared to L-amino acid-containingcounterparts. Thus, the construction of peptides, etc., incorporatingD-amino acids can be particularly useful when greater intracellularstability is desired or required. More specifically. D-peptides, etc.,are resistant to endogenous peptidases and proteases, thereby providingimproved bioavailability of the molecule, and prolonged lifetimes invivo when such properties are desirable. Additionally, D-peptides, etc.,cannot be processed efficiently for major histocompatibility complexclass II-restricted presentation to T helper cells, and are therefore,less likely to induce humoral immune responses in the whole organism.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

Also included in the invention are polynucleotides encoding polypeptidesof the antibodies. The term “polynucleotide” or “nucleotide sequence” isintended to indicate a consecutive stretch of two or more nucleotidemolecules. The nucleotide sequence may be of genomic, cDNA, RNA,semisynthetic or synthetic origin, or any combination thereof.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of ordinary skill inthe art will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine, and TGG, which isordinarily the only codon for tryptophan) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of ordinary skill in the art willrecognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are known to those of ordinary skill in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and allelesdescribed herein.

Conservative substitution tables providing functionally similar aminoacids are known to those of ordinary skill in the art. The followingeight groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Glycine (G); 2) Asparticacid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (1), Leucine (L), Methionine(M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see,e.g., Creighton, Proteins: Structures and Molecular Properties (W HFreeman & Co.; 2nd edition (December 1993)

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a percentage of amino acidresidues or nucleotides that are the same (i.e., about 60% identity,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, orabout 95% identity over a specified region), when compared and alignedfor maximum correspondence over a comparison window, or designatedregion as measured using one of the following sequence comparisonalgorithms (or other algorithms available to persons of ordinary skillin the art) or by manual alignment and visual inspection. Alignment forpurposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLEsoftware. This definition also refers to the complement of a testsequence. The identity can exist over a region that is at least about 50amino acids or nucleotides in length, or over a region that is 75-100amino acids or nucleotides in length, or, where not specified, acrossthe entire sequence of a polynucleotide or polypeptide. A polynucleotideencoding a polypeptide of the present invention, including homologs fromspecies other than human, may be obtained by a process comprising thesteps of screening a library under stringent hybridization conditionswith a labeled probe having a polynucleotide sequence described hereinor a fragment thereof, and isolating full-length cDNA and genomic clonescontaining said polynucleotide sequence. Such hybridization techniquesare well known to the skilled artisan.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are known to those of ordinary skill in the art. Optimalalignment of sequences for comparison can be conducted, including butnot limited to, by the local homology algorithm of Smith and Waterman(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Informationavailable at the World Wide Web at ncbi.nlm.nih.gov. The BLAST algorithmparameters W, T, and X determine the sensitivity and speed of thealignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength of 3, and expectation (E) of 10,and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands. The BLAST algorithm istypically performed with the “low complexity” filter turned off.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, or less than about0.01, or less than about 0.001.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (including but not limited to,total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to hybridizationof sequences of DNA, RNA, or other nucleic acids, or combinationsthereof under conditions of low ionic strength and high temperature asis known in the art. Typically, under stringent conditions a probe willhybridize to its target subsequence in a complex mixture of nucleic acid(including but not limited to, total cellular or library DNA or RNA) butdoes not hybridize to other sequences in the complex mixture. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).

As used herein, the terms “engineer, engineered, engineering”, areconsidered to include any manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpoly peptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches. The engineered proteins are expressed and produced bystandard molecular biology techniques.

By “isolated nucleic acid molecule or polynucleotide” is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated. Furtherexamples of an isolated polynucleotide include recombinantpolynucleotides maintained in heterologous host cells or purified(partially or substantially) polynucleotides in solution. An isolatedpolynucleotide includes a polynucleotide molecule contained in cellsthat ordinarily contain the polynucleotide molecule, but thepolynucleotide molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation. Isolated RNA molecules include in vivo or in vitro RNAtranscripts, as well as positive and negative strand forms, anddouble-stranded forms. Isolated polynucleotides or nucleic acidsdescribed herein, further include such molecules produced synthetically,e.g., via PCR or chemical synthesis. In addition, a polynucleotide or anucleic acid, in certain embodiments, includes a regulatory element suchas a promoter, ribosome binding site, or a transcription terminator.

The term “polymerase chain reaction” or “PCR” generally refers to amethod for amplification of a desired nucleotide sequence in vitro, asdescribed, for example, in U.S. Pat. No. 4,683,195. In general, the PCRmethod involves repeated cycles of primer extension synthesis, usingoligonucleotide primers capable of hybridizing preferentially to atemplate nucleic acid.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100) nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at the5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. As a practical matter,whether any particular polynucleotide sequence is at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of thepresent invention can be determined conventionally using known computerprograms, such as the ones discussed above for polypeptides (e.g.ALIGN-2).

A derivative, or a variant of a polypeptide is said to share “homology”or be “homologous” with the peptide if the amino acid sequences of thederivative or variant has at least 50% identity with a 100 amino acidsequence from the original peptide. In certain embodiments, thederivative or variant is at least 75% the same as that of either thepeptide or a fragment of the peptide having the same number of aminoacid residues as the derivative. In certain embodiments, the derivativeor variant is at least 85% the same as that of either the peptide or afragment of the peptide having the same number of amino acid residues asthe derivative. In certain embodiments, the amino acid sequence of thederivative is at least 90% the same as the peptide or a fragment of thepeptide having the same number of amino acid residues as the derivative.In some embodiments, the amino acid sequence of the derivative is atleast 95% the same as the peptide or a fragment of the peptide havingthe same number of amino acid residues as the derivative. In certainembodiments, the derivative or variant is at least 99% the same as thatof either the peptide or a fragment of the peptide having the samenumber of amino acid residues as the derivative.

The term “modified,” as used herein refers to any changes made to agiven polypeptide, such as changes to the length of the polypeptide, theamino acid sequence, chemical structure, co-translational modification,or post-translational modification of a polypeptide. The form“(modified)” term means that the polypeptides being discussed areoptionally modified, that is, the polypeptides under discussion can bemodified or unmodified.

In some aspects, a polypeptide comprises an amino acid sequence that isat least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%identical to a relevant (e.g., polypeptide and/or antibody) amino acidsequence or fragment thereof set forth in the Table(s) or accessionnumber(s) disclosed herein. In some aspects, an isolated antibody orprotein disclosed herein comprises an amino acid sequence encoded by apolynucleotide that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% identical to a relevant nucleotide sequence or fragmentthereof set forth in Table(s) or accession number(s) disclosed herein.In some aspects, a nucleotide sequence comprises a nucleotide sequencethat is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%identical to a nucleotide sequence disclosed herein such as those setforth in the Table(s) or accession number(s) disclosed herein.

Pharmaceutical Compositions

The present application provides compositions comprising the antibodiesincluding pharmaceutical compositions comprising any one or more of theantibodies described herein with one or more pharmaceutically acceptableexcipients. In some embodiments the composition is sterile. Thepharmaceutical compositions generally comprise an effective amount of anantibody.

These compositions can comprise, in addition to one or more of theantibodies disclosed herein, a pharmaceutically acceptable excipient,carrier, buffer, stabilizer or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material can depend on the route ofadministration, e.g. oral, intravenous, cutaneous or subcutaneous,nasal, intramuscular, intraperitoneal routes.

Pharmaceutical compositions for oral administration can be in tablet,capsule, powder or liquid form. A tablet can include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol can beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives can be included, as required.

Whether it is a polypeptide, antibody (e.g., anti-TREM2 antibody),nucleic acid, small molecule or other pharmaceutically useful compoundthat is to be given to an individual, administration is preferably in a“therapeutically effective amount” or “prophylactically effectiveamount” (as the case can be, although prophylaxis can be consideredtherapy), this being sufficient to show benefit to the individual. Theactual amount administered, and rate and time-course of administration,will depend on the nature and severity of protein aggregation diseasebeing treated. Prescription of treatment, e.g. decisions on dosage etc.,is within the responsibility of general practitioners and other medicaldoctors, and typically takes account of the disorder to be treated, thecondition of the individual subject, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

A composition can be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Methods

Methods of Preparation

Antibodies described herein can be produced using recombinant methodsand compositions, e.g., as described in U.S. Pat. No. 4,816,567.

In one embodiment, isolated nucleic acid encoding an antibody describedherein is provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody) or anamino acid sequence comprising the VHH of a single domain antibody. In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In one embodiment, thenucleic acid is provided in a multicistronic vector. In a furtherembodiment, a host cell comprising such nucleic acid is provided. In onesuch embodiment, a host cell comprises (e.g., has been transformedwith): (1) a vector comprising a nucleic acid that encodes an amino acidsequence comprising the VL of the antibody and an amino acid sequencecomprising the VH of the antigen-binding polypeptide construct, or (2) afirst vector comprising a nucleic acid that encodes an amino acidsequence comprising the VL of the antigen-binding polypeptide constructand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antigen-binding polypeptide construct.In one embodiment, the host cell is eukaryotic, e.g. a Chinese HamsterOvary (CHO) cell, or human embryonic kidney (HEK) cell, or lymphoid cell(e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making anantibody is provided, wherein the method comprises culturing a host cellcomprising nucleic acid encoding the antibody, as provided above, underconditions suitable for expression of the antibody, and optionallyrecovering the antibody from the host cell (or host cell culturemedium).

For recombinant production of the antibody, nucleic acid encoding anantibody, e.g., as described above, is isolated and inserted into one ormore vectors for further cloning and/or expression in a host cell. Suchnucleic acid may be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody).

The term “substantially purified” refers to a construct describedherein, or variant thereof that may be substantially or essentially freeof components that normally accompany or interact with the protein asfound in its naturally occurring environment, i.e. a native cell, orhost cell in the case of recombinantly produced heteromultimer that incertain embodiments, is substantially free of cellular material includespreparations of protein having less than about 30%, less than about 25%,less than about 20%, less than about 15%, less than about 10%, less thanabout 5%, less than about 4%, less than about 3%, less than about 2%, orless than about 1% (by dry weight) of contaminating protein. When theheteromultimer or variant thereof is recombinantly produced by the hostcells, the protein in certain embodiments is present at about 30%, about25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%,about 2%, or about 1% or less of the dry weight of the cells. When theheteromultimer or variant thereof is recombinantly produced by the hostcells, the protein, in certain embodiments, is present in the culturemedium at about 5 g/L, about 4 g/L, about 3 g/L, about 2 g/L, about 1g/L, about 750 mg/L, about 500 mg/L, about 250 mg/L, about 100 mg/L,about 50 mg/L, about 10 mg/L, or about 1 mg/L or less of the dry weightof the cells. In certain embodiments, “substantially purified”heteromultimer produced by the methods described herein, has a puritylevel of at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, specifically, a puritylevel of at least about 75%, 80%, 85%, and more specifically, a puritylevel of at least about 90%, a purity level of at least about 95%, apurity level of at least about 99% or greater as determined byappropriate methods such as SDS/PAGE analysis, RP-HPLC, SEC, andcapillary electrophoresis.

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.Host cells can include CHO, derivatives of CHO, NS0, Sp2O, CV-1,VERO-76, HeLa, HepG2, Per.C6, or BHK.

As used herein, the term “eukaryote” refers to organisms belonging tothe phylogenetic domain Eucarya such as animals (including but notlimited to, mammals, insects, reptiles, birds, etc.), ciliates, plants(including but not limited to, monocots, dicots, algae, etc.), fungi,yeasts, flagellates, microsporidia, protists, etc.

As used herein, the term “prokaryote” refers to prokaryotic organisms.For example, a non-eukaryotic organism can belong to the Eubacteria(including but not limited to, Escherichia coli, Thermus thermophilus,Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonasaeruginosa, Pseudomonas putida, etc.) phylogenetic domain, or theArchaea (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.)phylogenetic domain.

For example, antibody may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized.” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibodies arealso derived from multicellular organisms (invertebrates andvertebrates). Examples of invertebrate cells include plant and insectcells. Numerous baculoviral strains have been identified which may beused in conjunction with insect cells, particularly for transfection ofSpodoplera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N. Y. Acad. Si. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., ProcNatl. Acad. Sc. USA 77:4216 (1980)); and myeloma cell lines such as Y0,NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

In one embodiment, the antibodies described herein are produced instable mammalian cells, by a method comprising: transfecting at leastone stable mammalian cell with: nucleic acid encoding the antibody, in apredetermined ratio; and expressing the nucleic acid in the at least onemammalian cell. In some embodiments, the predetermined ratio of nucleicacid is determined in transient transfection experiments to determinethe relative ratio of input nucleic acids that results in the highestpercentage of the antibody in the expressed product.

In some embodiments is the method of producing an antibody in stablemammalian cells as described herein wherein the expression product ofthe at least one stable mammalian cell comprises a larger percentage ofthe desired glycosylated antibody as compared to the monomeric heavy orlight chain polypeptides, or other antibodies.

In some embodiments is the method of producing a glycosylated antibodyin stable mammalian cells described herein, said method comprisingidentifying and purifying the desired glycosylated antibody. In someembodiments, the said identification is by one or both of liquidchromatography and mass spectrometry.

If required, the antibodies can be purified or isolated afterexpression. Proteins may be isolated or purified in a variety of waysknown to those skilled in the art. Standard purification methods includechromatographic techniques, including ion exchange, hydrophobicinteraction, affinity, sizing or gel filtration, and reversed-phase,carried out at atmospheric pressure or at high pressure using systemssuch as FPLC and HPLC. Purification methods also includeelectrophoretic, immunological, precipitation, dialysis, andchromatofocusing techniques. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.As is well known in the art, a variety of natural proteins bind Fc andantibodies, and these proteins can find use in the present invention forpurification of antibodies. For example, the bacterial proteins A and Gbind to the Fc region. Likewise, the bacterial protein L binds to theFab region of some antibodies. Purification can often be enabled by aparticular fusion partner. For example, antibodies may be purified usingglutathione resin if a GST fusion is employed, Ni⁺² affinitychromatography if a His-tag is employed or immobilized anti-flagantibody if a flag-tag is used. For general guidance in suitablepurification techniques, see, e.g. incorporated entirely by referenceProtein Purification: Principles and Practice, 3^(nd) Ed., Scopes,Springer-Verlag, NY, 1994, incorporated entirely by reference. Thedegree of purification necessary will vary depending on the use of theantibodies. In some instances no purification is necessary.

In certain embodiments the antibodies are purified using Anion ExchangeChromatography including, but not limited to, chromatography onQ-sepharose, DEAE sepharose, poros HQ, poros DEAF, Toyopearl Q,Toyopearl QAE, Toyopearl DEAE, Resource/Source Q and DEAE, Fractogel Qand DEAE columns.

In specific embodiments the proteins described herein are purified usingCation Exchange Chromatography including, but not limited to,SP-sepharose, CM sepharose, poros HS, poros CM, Toyopearl SP, ToyopearlCM, Resource/Source S and CM, Fractogel S and CM columns and theirequivalents and comparables.

In addition, antibodies described herein can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W. H. Freeman & Co., N.Y. andHunkapiller et al., Nature, 310:105-111 (1984)). For example, apolypeptide corresponding to a fragment of a polypeptide can besynthesized by use of a peptide synthesizer. Furthermore, if desired,nonclassical amino acids or chemical amino acid analogs can beintroduced as a substitution or addition into the polypeptide sequence.Non-classical amino acids include, but are not limited to, to theD-isomers of the common amino acids, 2,4diaminobutyric acid, alpha-aminoisobutyric acid, 4aminobutyric acid, Abu, 2-amino butyric acid, g-Abu,e-Ahx, 6amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, alanine, fluoro-aminoacids, designer amino acids such as methyl amino acids, C-methyl aminoacids, N-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

Methods of Use

In one aspect, the present application provides methods of contactingnon-stimulatory myeloid cells with an anti-TREM2 antibody, such as ahuman antibody, which results in the disabling of the non-stimulatorymyeloid cells.

In another aspect, the present application provides methods ofcontacting non-stimulatory myeloid cells with an anti-TREM2 mouseantibody, which results in the disabling of the non-stimulatory myeloidcells.

In some embodiments the non-stimulatory cells are one or more of DC1cells, and TAM cells.

In some embodiments, the present application provides methods ofdisabling non-stimulatory myeloid cells, comprising contacting thenon-stimulatory myeloid cells with a TREM2 antibody, thereby killing thenon-stimulatory myeloid cells. Disabling refers to rendering a cellpartially or completely non-functional. In some embodiments, thedisabling of the non-stimulatory myeloid cells leads to inducing growtharrest in the cells. In some embodiments, the disabling of thenon-stimulatory myeloid cells leads to apoptosis in the cells. In someembodiments, the disabling of the non-stimulatory cells leads to lysisof the cells, as for example by complement dependent cytotoxicity (CDC)or antibody-dependent cell cytotoxicity (ADCC). In some embodiments, thedisabling of the non-stimulatory myeloid cells leads to necrosis in thecells. In some embodiments, the disabling of the non-stimulatory myeloidcells leads to inducing growth arrest in the cells. In some embodiments,the disabling of the non-stimulatory myeloid cells leads to inactivatingthe cells. In some embodiments, the disabling of the non-stimulatorymyeloid cells leads to neutralizing the activity of a TREM2 protein inthe cells. In some embodiments, the disabling of the non-stimulatorymyeloid cells leads to reduction in proliferation of the cells. In someembodiments, the disabling of the non-stimulatory myeloid cells leads todifferentiation of the cells. In some embodiments, the disabling of thenon-stimulatory myeloid cells leads to a decrease in the cells' abilityto act as inhibitory antigen presenting cells or leads to an increase inthe cells' ability to act as activating antigen-presenting cells. Insome embodiments, the disabling of the non-stimulatory myeloid cellsleads to the mislocalization of the cells within tumor tissue or tumormicroenvironment (TME). In some embodiments, the disabling of thenon-stimulatory myeloid cells leads to an altered spatial organizationof the cells within tumor tissue or tumor microenvironment. In someembodiments, the disabling of the non-stimulatory myeloid cells leads toan altered temporal expression of the cells within tumor tissue or TME.In some embodiments, the method further comprises removing thenon-stimulatory myeloid cells.

In any and all aspects of disabling non-stimulatory myeloid cells asdescribed herein, any increase or decrease or alteration of an aspect ofcharacteristic(s) or function(s) is as compared to a cell not contactedwith an anti-TREM2 antibody.

In another aspect, the present application provides methods ofcontacting non-stimulatory myeloid cells with an anti-TREM2 antibody,which results in the modulation of function of the non-stimulatorymyeloid cells. The modulation can be any one or more of the following.In some embodiments the non-stimulatory cells are one or more of DC1cells, TAM1 cells, and TAM2 cells. In some embodiments, the modulationof function leads to the disabling of non-stimulatory myeloid cells. Insome embodiments, the modulation of function of the non-stimulatorymyeloid cells leads to an increase in the cells' abilities to stimulateboth native and activated CD8+ T-cells, for example, by increasing theability of non-stimulatory cells to cross-present tumor antigen on MHCImolecules to naive CD8+ T-cells. In some embodiments, the modulationincreases the T-cell stimulatory function of the non-stimulatory myeloidcells, including, for example, the cells' abilities to trigger T-cellreceptor (TCR) signaling, T-cell proliferation, or T-cell cytokineproduction. In one embodiment, the survival of the non-stimulatory cellis decreased or the proliferation of the non-stimulatory cell isdecreased. In one embodiment, the ratio of stimulatory myeloid cells tonon-stimulatory myeloid cells is increased.

In any and all aspects of decreasing the function of non-stimulatorymyeloid cells as described herein, any increase or decrease oralteration of an aspect of characteristic(s) or function(s) is ascompared to a cell not contacted with an anti-TREM2 antibody.

In some embodiments, the present application provides methods of killing(also referred to as inducing cell death) non-stimulatory myeloid cells,comprising contacting the non-stimulatory myeloid cells with ananti-TREM2 antibody, thereby killing the non-stimulatory myeloid cells.In some embodiments the killing is increased relative to non-stimulatorymyeloid cells that have not been contacted with an anti-TREM2 antibody.In some embodiments, the contacting induces apoptosis in thenon-stimulatory myeloid cells. In some embodiments, the contactinginduces apoptosis in the non-stimulatory myeloid cells. In someembodiments, the non-stimulatory myeloid cells are in a population ofimmune cells comprising non-stimulatory myeloid cells and stimulatorymyeloid cells. In some embodiments, the method further comprisesremoving the non-stimulatory myeloid cells. In some embodiments, 10%-80%of the cells are killed. In some embodiments, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, or 80% of the cells are killed.

In some embodiments, the present application provides methods ofincreasing the ratio of stimulatory myeloid cells to non-stimulatorymyeloid cells in a population of immune cells comprising stimulatorymyeloid cells and non-stimulatory myeloid cells, comprising contactingthe population of immune cells with an anti-TREM2 antibody. In someembodiments the ratio is increased relative to a population of cellsthat have not been contacted with an anti-TREM2 antibody. In someembodiments the ratio of DC2 cells to DC1 cells is increased. In someembodiments the ratio of DC2 cells to TAM1 cells is increased. In someembodiments the ratio of DC2 cells to TAM2 cells is increased. In someembodiments the ratio of DC2 cells to TAM1+TAM2 cells is increased. Insome embodiments the ratio of DC2 cells to TAM1+DC cells is increased.In some embodiments the ratio of DC2 cells to DC1+TAM2 cells isincreased. In some embodiments the ratio of DC2 cells to DC1+TAM1+TAM2cells is increased. In some embodiments, at least the ratio is increasedby 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

In some embodiments the ratio of stimulatory myeloid cells tonon-stimulatory myeloid cells prior to contacting ranges from0.001:1-0.1:1. In some embodiments the ratio of stimulatory myeloidcells to non-stimulatory myeloid cells following the contacting rangesfrom 0.1:1-100:1.

In some embodiments, the non-stimulatory myeloid cells are reduced innumber. In some embodiments the stimulatory myeloid cells are DC2 cells.In some embodiments, the non-stimulatory myeloid cells are killed, forexample by necrosis, or apoptosis. In some embodiments, thenon-stimulatory myeloid cells are induced to undergo growth arrest. Insome embodiments the non-stimulatory myeloid cells no longerproliferate. In some embodiments the spatial localization of thenon-stimulatory myeloid cells is altered, and the ratio is increased ina particular region of the TME. In some embodiments the temporalexpression of the non-stimulatory myeloid cells is altered, and theratio is increased during a particular time during the development ofthe tumor.

In some embodiments, the contacting is in vitro. In some embodiments,the contacting is in vivo. In some particular embodiments, thecontacting is in vivo in a human. In some embodiments, the contacting iseffected by administering an anti-TREM2 antibody. In some embodiments,the individual receiving the antibody (such as a human) has cancer.

In another aspect, the invention provides methods of treating animmune-related condition (e.g., cancer) in an individual comprisingadministering to the individual an effective amount of a compositioncomprising an anti-TREM2 antibody. In another aspect, the inventionprovides methods of enhancing an immune response in an individualcomprising administering to the individual an effective amount of acomposition comprising an anti-TREM2 antibody. In some embodiments thesemethods are further provided in combination with other co-therapies suchas a PDL blockade therapy, anti-PD-1 antibodies, anti-PD-L1 antibodies,anti-PD-L2 antibodies, a CTLA4 blockade therapy, anti-CTLA-4 antibodies,generalized checkpoint blockade therapy in which inhibitory molecules onT cells are blocked, adoptive T-cell therapy, CAR T-cell therapy,dendritic cell or other cellular therapies, as well as conventionalchemotherapies.

In some embodiments, the method further comprises determining theexpression level of TREM2 protein in a biological sample from theindividual. In some embodiments the biological sample includes, but isnot limited to a body fluid, a tissue sample, an organ sample, urine,feces, blood, saliva, CSF and any combination thereof. In someembodiments the biological sample is derived from a tumor tissue. Insome embodiments, the expression level comprises the mRNA expressionlevel of mRNA encoding TREM2 protein. In some embodiments, theexpression level of TREM2 protein comprises the protein expression levelof NSM. In some embodiments the expression level of TREM2 protein isdetected in the sample using a method selected from the group consistingof FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry,immunofluorescence, radioimmunoassay, dot blotting, immunodetectionmethods, HPLC, surface plasmon resonance, optical spectroscopy, massspectrometery. HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq,microarray analysis, SAGE, MassARRAY technique, and FISH, andcombinations thereof.

In another aspect, the present application provides methods fordetermining the presence or absence of non-stimulatory myeloid cells ingeneral, or for determining the presence or absence of particularnon-stimulatory myeloid cells (for example DC1 cells, TAM1 cells, and/orTAM2 cells) comprising: contacting a population of cells comprisingnon-stimulatory myeloid cells with an anti-TREM2 antibody; andquantifying the number non-stimulatory myeloid cells. In another aspect,the present application provides methods for determining the presence orabsence of non-stimulatory myeloid cells comprising: contacting apopulation of immune cells comprising non-stimulatory myeloid cells andstimulatory myeloid cells with an anti-TREM2 antibody; detecting acomplex or moiety indicating the binding of the antibody to the cell andoptionally quantifying the number of non-stimulatory myeloid cells inthe population. In another aspect, methods of determining the relativeratio of non-stimulatory myeloid cells to stimulatory myeloid cells areprovided, comprising: contacting a population of immune cells comprisingnon-stimulatory myeloid cells and stimulatory myeloid cells with ananti-TREM2 antibody; quantifying the number of stimulatory myeloid cellsand non-stimulatory myeloid cells; and determining the relative ratio ofnon-stimulatory myeloid cells to stimulatory myeloid cells.

In embodiments described herein for detection and/or quantification, ananti-TREM2 antibody binds to a TREM2 protein, but does not necessarilyhave to affect a biological response, such as ADCC, although it may havean effect on a biological response.

In another aspect, the present invention provides methods foridentifying an individual who may respond to immunotherapy (e.g. with ananti-TREM2 antibody) for the treatment of an immune-related condition(e.g. cancer) comprising: detecting the expression level of TREM2protein in a biological sample from the individual; and determiningbased on the expression level of TREM2 protein, whether the individualmay respond immunotherapy, wherein an elevated level of TREM2 protein inthe individual relative to that in a healthy individual indicates thatthe individual may respond to immunotherapy. In some embodiments, thesemethods may also be used for diagnosing an immune-related condition(e.g. cancer) in the individual and are based the expression level ofTREM2 protein, wherein an elevated level of TREM2 protein in theindividual relative to that in a healthy individual indicates that theindividual suffers from cancer. In some embodiments, the expressionlevel comprises the mRNA expression level of mRNA encoding TREM2protein.In other embodiments, the expression level of TREM2 protein comprisesthe protein expression level of TREM2 protein. In some embodiments theexpression level of TREM2 protein is detected in the sample using amethod selected from the group consisting of FACS, Western blot, ELISA,immunoprecipitation, immunohistochemistry, immunofluorescence,radioimmunoassay, dot blotting, immunodetection methods, HPLC, surfaceplasmon resonance, optical spectroscopy, mass spectrometery, HPLC, qPCR,RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE.MassARRAY technique, and FISH, and combinations thereof. In theseembodiments, an anti-TREM2 antibody binds to the TREM2 protein, but doesnot necessarily have to affect a biological response, such as ADCC. Insome embodiments the biological sample is derived from a tumor tissue.In some embodiments the biological sample includes, but is not limitedto a body fluid, a tissue sample, an organ sample, urine, feces, blood,saliva, CSF and any combination thereof.

Also disclosed herein is a method of enhancing a subject immune responseto tumors or enhancing the efficacy of immunotherapy treatments. Ingeneral, a treatment that increases the abundance of SDC's will improvesubject outcome, such as recurrence-free survival time, and will enhancethe efficacy of cancer immunotherapy treatments. A treatment canincrease the relative or absolute abundance of SDC cells in a subject'stumor. A treatment can decrease the relative or absolute abundance ofNSM cells in a subject's tumor.

Exemplary methods of the general treatment strategy include increasingthe numbers of SDC's by systemic introduction of Flt3L. Another methodis treatment of a subject's autologous bone-marrow or blood cells withFlt3L while simultaneously blocking CSF1. Expression, for example byretrovirus, of SDC transcription factors such as IRF8, Myc11 or BATF3 orZBTB46 in bone-marrow or blood progenitor populations may also be usedto drive SDC development. Another strategy of treatment includes thesystematic elimination of NSM cells while selectively sparing the SDC.This can generate an overall favorable change in the ratio of thesepopulations. Elimination of NSM cells may be accomplished by any means,including the administration (systemic or localized to the tumor) ofantibodies against TREM2 surface proteins.

In some embodiments, SDC-enhancing treatments are applied as atherapeutic treatment to better enable the subject's native immunesystem in controlling or eradicating the cancer. In another embodiment,the SDC-enhancing treatments of the invention are applied in combinationwith a therapeutic treatment such as an immunotherapy treatment (suchapplication being prior to, concurrent with, or after the immunotherapytreatment) wherein the SDC-enhancing treatment acts as an accessory oradjuvant treatment to increase the efficacy of the therapeutictreatment.

Methods of Administration

In some embodiments, the methods provided herein are useful for thetreatment of an immune-related condition in an individual. In oneembodiment, the individual is a human and the antibody is a TREM2antibody. In another embodiment, the individual is a mouse and theantibody is a TREM2 antibody.

In some embodiments, for in vivo administration of the anti-TREM2antibodies described herein, normal dosage amounts may vary from about10 ng/kg up to about 100 mg/kg of an individual's body weight or moreper day, preferably about 1 mg/kg/day to 10 mg kg/day, depending uponthe route of administration. For repeated administrations over severaldays or longer, depending on the severity of the disease or disorder tobe treated, the treatment is sustained until a desired suppression ofsymptoms is achieved. An exemplary dosing regimen comprisesadministering an initial dose of an anti-TREM2 antibody of about 2mg/kg, followed by a weekly maintenance dose of about 1 mg/kg everyother week. Other dosage regimens may be useful, depending on thepattern of pharmacokinetic decay that the physician wishes to achieve.For example, dosing an individual from one to twenty-one times a week iscontemplated herein. In certain embodiments, dosing ranging from about 3μg/kg to about 2 mg/kg (such as about 3 μg/kg, about 10 μg/kg, about 30μg/kg, about 100 μg/kg, about 300 μg/kg, about 1 mg/kg, and about2/mg/kg) may be used. In certain embodiments, dosing frequency is threetimes per day, twice per day, once per day, once every other day, onceweekly, once every two weeks, once every four weeks, once every fiveweeks, once every six weeks, once every seven weeks, once every eightweeks, once every nine weeks, once every ten weeks, or once monthly,once every two months, once every three months, or longer. Progress ofthe therapy is easily monitored by conventional techniques and assays.The dosing regimen, including the anti-TREM2 antibody administered, canvary over time independently of the dose used.

In some embodiments, the methods provided herein (such as methods ofenhancing an immune response or effecting the disabling ofnon-stimulatory myeloid cells) are useful for the treatment of cancerand as such an individual receiving an anti-TREM2 antibody or ananti-TREM2 antibody has cancer.

Any suitable cancer may be treated with the antibodies provided herein.The cancer can be any carcinoma, adenocarcinoma, soft tissue, sarcoma,teratomas, melanoma, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma,or brain cancer known in the medical field. In some embodiments, thecancer is a solid cancer. In some embodiments, the cancer is a liquidcancer. In some embodiments, the cancer is imnunoevasive. In someembodiments, the cancer is immunoresponsive. In some embodiments, thecancer is melanoma, kidney, hepatobiliary, head-neck squamous carcinoma(HNSC), pancreatic, colon, bladder, glioblastoma, prostate, lung, breast(mammary), ovarian, gastric, kidney, bladder, esophageal, renal,melanoma, leukemia, lymphoma, or mesothelioma. In some embodiments, thecancer is colon cancer, pancreatic cancer, or breast cancer.

In some embodiments the immune-related condition is an immune-relatedcondition associated with the expression of TREM2 protein onnon-stimulatory myeloid cells (in humans) or the expression of a homologof TREM2 protein in a non-human species. In some embodiments theimmune-related condition is an immune-related condition associated withthe overexpression of TREM2 protein on non-stimulatory myeloid cells, ascompared to stimulatory myeloid-cells. In some embodiments theoverexpression of the TREM2 mRNA or the TREM2 protein is about at least2 fold, 5 fold, 10 fold, 25 fold, 50 fold, or 100 fold higher ascompared to stimulatory myeloid cells.

In some embodiments, the treatment enhances an immune response in thesubject. In some embodiments, the enhanced immune response is anadaptive immune response. In some embodiments, the enhanced immuneresponse is an innate immune response.

In some embodiments, an antibody is administered intravenously,intramuscularly, subcutaneously, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally. An effective amountof an anti-TREM2 antibody may be administered for the treatment ofcancer. The appropriate dosage of the anti-TREM2 antibody may bedetermined based on the type of cancer to be treated, the type of theanti-TREM2 antibody, the severity and course of the cancer, the clinicalcondition of the individual, the individual's clinical history andresponse to the treatment, and the discretion of the attendingphysician.

Combination Therapies

In some embodiments, an antibody provided herein is administered with atleast one additional therapeutic agent. Any suitable additionaltherapeutic agent may be administered with an antibody provided herein.In some embodiments, the immunotherapy selected from a checkpointinhibitor; a checkpoint inhibitor of T cells; anti-PD1 antibody;anti-PDL1 antibody; anti-CTLA4 antibody; adoptive T cell therapy; CAR-Tcell therapy; a dendritic cell vaccine; a monocyte vaccine; an antigenbinding protein that binds both a T cell and an antigen presenting cell;a BiTE dual antigen binding protein; a toll-like receptor ligand; acytokine; a cytotoxic therapy; a chemotherapy; a cytostatic agent; aradiotherapy; a small molecule inhibitor; a small molecule agonist; animmunomodulator; and an epigenetic modulator, an combinations thereof.

In some embodiments, the additional therapeutic agent is an antibody. Insome embodiments, the additional therapeutic agent is an antibody thatbinds a protein or proteins on a tumor cell surface.

For the treatment of cancer, the anti-TREM2 antibody may be combinedwith one or more antibodies that inhibit immune checkpoint proteins. Ofparticular interest are immune checkpoint proteins displayed on thesurface of a tumor cell. The immune-checkpoint receptors that have beenmost actively studied in the context of clinical cancer immunotherapy,cytotoxic T-lymphocyte-associated antigen 4 (CTLA4; also known as CD152)and programmed cell death protein 1 (PD1; also known as CD279), are bothinhibitory receptors. The clinical activity of antibodies that blockeither of these receptors implies that antitumor immunity can beenhanced at multiple levels and that combinatorial strategies can beintelligently designed, guided by mechanistic considerations andpreclinical models.

The two ligands for PD-1 are PD-1 ligand 1 (PD-L1; also known as B7-H1and CD274) and PD-L2 (also known as B7-DC and CD273). PD-L1 is expressedon cancer cells and through binding to its receptor PD-1 on T cells itinhibits T cell activation/function. Inhibitors that block theinteraction of PD-1 with its cognate ligands on the cancer cells. PD-L1and PD-L2, can result in both increased T cell activation and function,and prevent cancer cells from evading the immune system.

In some embodiments, the immunotherapy is an agent that interferes withPD-1 and PD-L1 or PD-L2 binding. In some embodiments, the immunotherapyis an anti-PD1 antibody. In some embodiments, the immunotherapy is ananti-PD-L1 antibody. In some embodiments, the immunotherapy is ananti-PD-L2 antibody.

Various PD-1, PD-L1, and PD-L2 antibodies are known in the art. In someembodiments, the additional therapeutic agent is at least one of:Atezolizumab (PD-L1), Avelumab (PD-L1), Durvalumab (PD-L1), Nivolumab(PD-1), Pembrolizumab (PD-1), Cemiplimab (PD-1), Ipilimumab (CTLA-4).Tremelimumab (CTLA-4), or any combination thereof.

The additional therapeutic agent can be administered by any suitablemeans. In some embodiments, an antibody provided herein and theadditional therapeutic agent are included in the same pharmaceuticalcomposition. In some embodiments, an antibody provided herein and theadditional therapeutic agent are included in different pharmaceuticalcompositions.

In embodiments where an antibody provided herein and the additionaltherapeutic agent are included in different pharmaceutical compositions,administration of the antibody can occur prior to, simultaneously,and/or following, administration of the additional therapeutic agent. Insome embodiments, administration of an antibody provided herein and theadditional therapeutic agent occur within about one month of each other.In some embodiments, administration of an antibody provided herein andthe additional therapeutic agent occur within about one week of eachother. In some embodiments, administration of an antibody providedherein and the additional therapeutic agent occur within about one dayof each other. In some embodiments, administration of an antibodyprovided herein and the additional therapeutic agent occur within abouttwelve hours of each other. In some embodiments, administration of anantibody provided herein and the additional therapeutic agent occurwithin about one hour of each other.

Kits and Articles of Manufacture

The present application provides kits comprising any one or more of theantibody compositions described herein. In some embodiments, the kitsfurther contain a component selected from any of secondary antibodies,reagents for immunohistochemistry analysis, pharmaceutically acceptableexcipient and instruction manual and any combination thereof. In onespecific embodiment, the kit comprises a pharmaceutical compositioncomprising any one or more of the antibody compositions describedherein, with one or more pharmaceutically acceptable excipients.

The present application also provides articles of manufacture comprisingany one of the antibody compositions or kits described herein. Examplesof an article of manufacture include vials (including sealed vials).

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1: Humanization of Anti-TREM2 Antibody Humanization of Clone#237920

A monoclonal Rat IgG2B Clone #237920 (R&D Systems Cat # MAB17291)specific for mouse and human TREM2 was used for sequence determinationand humanization. In brief, disulfide bonds in the antibody were reducedwith dithiothreitol (DTT) and free sulfhydryl groups were alkylated withiodoacetamide. The alkylated antibody was digested with sequencing-gradeendoproteinases, purified using spin columns, and sequence wasdetermined by LC-MS/MS analysis. The sequences are shown below.

SEQ ID NO Name Sequence 33 Rat IgG2B EVQLVESGGG LVQPGRSLKL clone #SCAASGFTFS NYYMAWVRQA 237920 PTKGLEWVAS LTNSGGSTYY heavy chainRDSVKGRFTL SRDNAKSTLY LQMDSLRSED TATYYCTREW AGSGYFDYWG QGVMVTVSSAQTTAPSVYPL APGCGDTTSS TVTLGCLVKG YFPEPVTVTW NSGALSSDVH TFPAVLQSGLYTLTSSVTSS TWPSQTVTCN VAHPASSTKV DKKVERRDGG IGHKCPTCPT CHKCPVPELLGGPSVFLFPP KPKDILLLSQ NAKVTCVVVD VSEEEPDVQF SWFVNNVEVH TAQTQPREEQYNSTFRVVSA LPLQHQDWMS GKEFKCKVNN KALPSPIEKT LSKPKGLVRK PQVYVMGPPTEQLTEQTVSL TCLTSGFLPN DIGVEWTSNG HIEKNYKNTE PVMDSDGSFF MYSKLNVERSRWDSRAPFVC SVVHEGLHNH HVEKSLSRPP G 34 Rat IgG2B NIVMTQSPKS MSLSVGDRVTclone # MNCKASQNVG NNLAWYQQKP 237920 GQSPKLLLYY TSNRFTGVPD light chainRFTGGGYGTD FTLTINSVQA EDAAFYYCQR IYNSPWTFGG GTKLELKRAD AAPTVSIFPPSTEQLATGGA SVVCLMNNFY PRDISVKWKI DGTERRDGVL DSVTDQDSKD STYSMSSTLSLTKADYESHN LYTCEVVHKT SSSPVVKSFN RNEC

The VH and VL sequences were compared to libraries of known humangermline sequences on the NCBT website(http://www.ncbi.nlmnih.gov/igblast/; Ye, J. et al. Nucleic AcidsResearch 41:W34-W40 (2013)). The databases used were IMGT human VH genes(F+ORF, 273 germline sequences) and IMGT human VLkappa genes (F+ORF, 74germline sequences).

For 237920 VH, human germline IGHV3-23(allele 1) was chosen as theacceptor sequence and the human heavy chain IGHJ4(allele 1) joiningregion (J gene) was chosen from human joining region sequences compiledat IMGT® the international ImMunoGeneTics information System®www.imgt.org (founder and director: Marie-Paule Lefranc, Montpellier,France).

For 237920 VL, human germline IGKV1-39(allele 1) was chosen as theacceptor sequence and human light chain IGKJ2(allele 1) joining region(J gene) was chosen from human joining region sequences compiled atIMGT® the international ImMunoGeneTics information System® www.imgt.org(founder and director: Marie-Paule Lefranc, Montpellier, France).

CDRs were defined according to the AbM definition (see the website ofDr. Andrew C. R. Martin www.bioinf.org.uk/abs/ for a table comparing CDRdefinitions). Alteration of human germline framework (i.e., non-CDRresidues in VH and VL) positions to corresponding parental murinesequence were used, e.g., to optimize binding of the humanized antibody.

Table 1A shows VL, VH, and full heavy and light chain sequences of thehumanized versions of mAb 237920 that were created. 37017 is the parenthumanized clone from which the other humanized versions were created viaadditional mutations. Table 1B shows the CDR sequences.

TABLE 1A SEQ ID NO Name Sequence  1 37012_VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREWAGSGYFDYWGQGTLVTVSS  2 37012_VLDIQMTQSPSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNRFTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIK  3 37013_VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREWAGSGYFDYWGQGTLVTVSS  4 37013_VLDIQMTQSPSSLSASVGDRVTMTCKASQNVGNNLAWYQQKPGKAPKLLLYYTSNRFTGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCQRIYNSPWTFGQGTKLELK  5 37014_VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVASLTNSGGSTYYADSVKGRFTLSRDNSKNTLYLQMNSLRAEDTAVYYCTREWAGSGYFDYWGQGTLVTVSS  6 37014_VLDIQMTQSPSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNRFTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIK  7 37017_VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEWAGSGYFDYWGQGTLVTVSS  8 37017_VLDIQMTQSPSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNRFTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIK 25 FullEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNSGGSTYY 37012_HADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREWAGSGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 26 FullDIQMTQSPSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNRFTGVPS 37012_LRFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 27 FullEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNSGGSTYY 37013_HADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREWAGSGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 28 FullDIQMTQSPSSLSASVGDRVTMTCKASQNVGNNLAWYQQKPGKAPKLLLYYTSNRFTGVPS 37013_LRFSGSGSGTDFTLTISSVQPEDFATYYCQRIYNSPWTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 29 FullEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVASLTNSGGSTYY 37014_HADSVKGRFTLSRDNSKNTLYLQMNSLRAEDTAVYYCTREWAGSGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 30 FullDIQMTQSPSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNRFTGVPS 37014_LRFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 31 FullEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNSGGSTYY 37017_HADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEWAGSGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 32 FullDIQMTQSPSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNRFTGVPS 37017_LRFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

TABLE 1B CDRs of humanized antibodies Table 1B CDR Sequence SEQ ID NOCDR-H1 FSNYYMA  9 CDR-H2 SLTNSGGSTY 10 CDR-H3 EWAGSGY 11 CDR-L1 NVGNNLA12 CDR-L2 YTSNRFT 13 CDR-L3 RIYNSPW 14

Alignment of the Framework of the Humanized Antibodies

CDR-H1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNSGGSTYY  60CDR-H2 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNSGGSTYY  60CDR-H3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWV A SLTNSGGSTYY 60 3-23*01 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYY 60 CDR-H1 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEWAGSGYFDYWGQGTLVTVSS119 CDR-H2 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TR EWAGSGYFDYWGQGTLVTVSS119 CDR-H3 ADSVKGRFTLSRDNSKNTLYLQMNSLRAEDTAVYYC TR EWAGSGYFDYWGQGTLVTVSS119 3-23*01 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK----------WGQGTLVTVSS109 CDR-L1 DIQMTQSPSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNRFTGVPS 60 CDR-L2 DIQMTQSPSSLSASVGDRVTMTCKASQNVGNNLAWYQQKPGKAPKLLLYYTSNRFTGVPS 60 1-39*01 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS 60 CDR-L1 RFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIK 107 CDR-L2RFSGSGSGTDFTLTISSVQPEDFATYYCQRIYNSPWTFGQGTKLELK 107 1-39*01RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP-PFGQGTKLEIK 106

In the VL domain, in CDRs, Asn28, Asn1, Asn32 and Asn53 have a lowpotential for deamidation based on sequence and conformaion. Asn93 has alow to medium potential for deamidation and could show a low level ofthis post-translational modification. In the VH domain, Asn31 has a lowpotential for deamidation based on sequence and conformation. In CDR-H2Asn53 has a medium potential for deamidation; to preventpost-translational modification, Asn53 could be altered to Gln, Ser orAla and maintenance of binding determined experimentally. In CDR-H3Trp100 may be solvent-exposed and have potential for oxidation,especially under stress conditions.

In-Solution Endoproteinase Digestion

In-solution endoproteinase digestions of the monoclonal antibody (mAb)were performed for mAb sequencing analysis. 50 μg of the antibody wasreduced with DTT, alkylated using iodoacetamide, acetone precipitatedand reconstituted in water at a concentration of 1 μg/μL. In-solutiondigestion of the antibody sample was performed by using 5 individualenzyme digestions: Asp-N, Chymotrypsin, Elastase, Trypsin and Pepsinfollowing manufacturer's instructions. Samples were then lyophilized,resuspended in 0.1% TFA and purified using a C18 Zip-Tip. Samples werethen dried by vacuum centrifugation and kept frozen until massspectrometry analysis.

Mass Spectrometry

Intact Mass Measurement

The mAb sample was denatured, reduced, and acidified. The proteins werethen analyzed using an Agilent 1100 HPLC connected to a Waters QToFUltima Global mass spectrometer (LC-ESI-TOF MS). The appropriate LC-MSspectra were processed (combined, subtracted, smoothed and deconvoluted)using Waters MassLynx 4.1 software.

LC-MS/MS Analysis

The purified peptides were re-suspended in 0.1% formic acid and one halfof each of the digests were analyzed on an Orbitrap analyzer(Q-Exactive, Thermo Fisher Scientific) outfitted with a nanospray sourceand EASY-nLC 1 (000 system (Thenno Fisher Scientific). Peptides wereloaded onto a 50 cm (75 μm inner diameter) EASY-Spray column packed withPepMap® RSLC 2 μm C18 resin (Thermo Fisher Scientific) at a pressure of800 Bar. Peptides were eluted at a rate of 250 nl/min using a gradientset up as 0%-30% acetonitrile in 0.1% formic acid over 60 min. Peptideswere introduced by nano-electrospray ion source into the Q-Exactive massspectrometer (Thermo Fisher Scientific). The instrument method consistedof one MS full scan (400-1600 m/z) in the Orbitrap mass analyzer with anautomatic gain control (AGC) target of 1E6, maximum ion injection timeof 120 ms and a resolution of 70 000 followed by 10 data-dependent MS/MSscans with a resolution of 17 500, an AGC target of 5E5, maximum iontime of 100 ms, and one microscan. The intensity threshold to trigger aMS/MS scan was set to an underfill ratio of 1.0%. Fragmentation occurredin the HCD collision cell with normalized collision energy set to 30.The dynamic exclusion was applied using a setting of 8 seconds.

Table 2 summarizes the biophysical characteristics of the humanizedclones. Molecular Weight and Extinction Coefficient are estimated forthe sum of the contributing protein chains in the quaternary structure.By default the calculation assumes equal and monomeric contribution fromeach chain. Extinction Coefficient is the predicted absorbance at 280 nmper molar protein in units of M⁻¹ cm⁻¹. Potential post-translationalmodifications such as glycosylation, phosphorylation, and proteolysisare not considered in Molecular Weight or Extinction Coefficientestimates.

TABLE 2 Extinction Molecular Isoelectric Antibody Coefficient Weight(Da) Point Titer (mg/L) PI37012 226380 145004 8.41 255.9 PI37013 226380145012 8.41 249.7 PI37014 226380 144972 8.41 259.9 PI37017 226380 1448708.45 198.61

Example 2: Production and Characterization of Anti-TREM2 AntibodiesAntibody Production and Characterization

Standard protein expression vectors were transfected into HEK293 usingstandard methods following which cells were grown for 7 days andharvested. In addition to HEK293, antibodies were also produced in 293cells that were made deficient in mammalian a1,6-fucosyltransferase(FUT8) by CRISPR/Cas9 editing (Alexander Weiss, University of Toronto).Supernatant pH was adjusted with 1M Hepes pH 7.4 and sodium azide wasadded to prevent microbial growth. KanCap A resin was used to captureproteins and the antibodies were eluted with 50 mM Citrate pH 3.5, 100mM NaCL after washing with PBS and PBS containing 1M sodium chloride.Immediately after elution, the solution was neutralized with 1M Tris (pH8) containing 0.5M Arginine. Biophysical characterization was conductedon protein that was buffer exchanged to PBS using standard techniques.Protein was quantified by OD280, quantity and concentration wasdetermined using calculated extinction coefficient. Reduced andnon-reduced SDS-PAGE (Biorad criterion Tris/Glycine/SDS, 4-20%) orPerkin Elmer GXII capillary electrophoresis system, was used todetermine purity and approximate molecular mass. Aggregation status wasdetermined by HPLC, with detection at 280 nm using a Sepax Zenix-CSEC-300, 3 um, 300 Å, 4.6*150 mm size exclusion column and PBS runningbuffer.

Antibody Affinity Measurement Using Surface Plasmon Resonance (SPR)

Binding kinetics were determined by surface plasmon resonance using aBiacore T200 (GE Healthcare, UK) with human TREM2 His (Sino Biological,Beijing, P.R. China) or human captured on Series S CM5 chips throughanti-His capture or TREM2 human IgG1 Fc fusion protein (in-house SECpurified to >95% purity) directly immobilized to chips by aminecoupling. Serial dilutions of indicated antibodies were injected at 30ul/minute for 2 minutes. PBS or system buffer was then injected at 30ul/minute for 400 seconds to observe dissociation. Binding responseswere corrected by subtraction of responses on a blank flow cell. Forkinetic analysis, a 1:1 Langmuir model of global fittings of k_(on) andk_(off) values, was used. The K_(d) values were determined from theratios of k_(on) and k_(off).

Table 3 shows antibody binding affinity to human TREM2-His measured bySPR.

TABLE 3 Rmax Chi² Antibody ka (1/Ms) kd (1/s) KD (M) (RU) (RU²) PI370121.70E+06 8.69E−03 5.12E−09 30.2053 1.1785 PI37013 1.14E+06 5.49E−034.82E−09 33.8282 1.3772 PI37014 5.11E+05 2.43E−03 4.74E−09 34.53631.3754

Table 4 shows antibody binding affinity to human TREM2-Fc measured bySPR.

TABLE 4 Rmax Antibody ka (1/Ms) kd (1/s) KD (M) (RU) Chi² (RU²) PI370124.96E+05 9.56E−04 1.93E−09 185.50 16.25 Afuc 4.47E+05 8.84E−04 1.98E−09174.90 13.83 PI37012 PI37013 5.17E+05 8.88E−04 1.72E−09 190.63 19.07PI37014 4.71E+05 7.32E−04 1.55E−09 184.88 17.65 PI37017 3.40E+056.14E−03 1.80E−08 35.05 4.58

At low ligand density (RL=500 RU), PI37017 binding kinetics to humanTREM2-Fc did not result in a good fit. This data indicates that the Apresent at position 97 and the K present at position 98 of the sequenceof SEQ ID NO:31 (clone 37017) likely causes a substantial loss of humanTREM2 binding upon humanization of the rat IgG2s. Clone #237920.Mutation of these framework residues (A97T and K98R) results inincreased human TREM2 binding by the humanized clones. See, for example,clone 37012.

Example 3: Cellular Binding of Anti-TREM2 Antibodies Cellular Binding(EC50 Measurement):

100,000 to 500,000 Expi 293 parental cells or Expi 293 cellsover-expressing human or mouse TREM2 were plated in 96 well plates anddead cells were stained with Zombie Near Infrared (Biolegend).Titrations of indicated unconjugated antibodies were incubated withthese cells within a range of 0 ug/ml to 10 ug/ml in a 1:3 dilutionrange across 8-10 points. Dependent on their isotype (hIgG1 or mIgG2a),these primary unconjugated antibodies were detected with Alexa Fluor 647conjugated anti-human Fc or anti-mouse Fc secondary antibodies (JacksonImmunoresearch). Alexa Fluor 647 signal was measured by flow cytometry(BD Fortessa X-14, BD Biosciences). EC50 values were calculated by curvefitting signal generated from antibodies binding to over-expressingcells over background fluorescence generated from HEK293 parental cellsin Graphpad Prism (Graphpad Software).

This data indicates that the A present at position 97 and the K presentat position 98 of the sequence of SEQ ID NO:31 (clone 37017) likelycauses a substantial loss of human TREM2 binding upon humanization ofthe rat IgG_(2B) Clone #237920. Mutation of these framework residues(A97T and K98R) results in increased human TREM2 binding by thehumanized clones. See, for example, clone 37012.

Table 5 shows half-maximal saturation binding of anti-TREM2 antibodiesto cell surface TREM2.

TABLE 5 Antibody Cell Line EC50 (nM) 237920 Expi-mTREM2 0.9 237920Expi-hTREM2 0.6 PI37012 Expi-mTREM2 0.5 PI37012 Expi-hTREM2 1.3 PI37013Expi-mTREM2 1.2 PI37013 Expi-hTREM2 1.4 PI37013 Expi-mTREM2 1.2 PI37014Expi-hTREM2 1.4 PI37017 Expi-mTREM2 3.6 PI37017 Expi-hTREM2 23.3

Example 4: PI-7012 Improves Anti-Tumor Activity in Combination withAnti-PD-1 Materials and Methods

CT26.WT (CRL-2638) cells were purchased from the American Type CultureCollection (ATCC). Antibodies for in vivo use were all tested forendotoxin and used at or below 0.2 EU/mg protein. The amino acidsequence of the anti-mouse PD-1 antibody from clone RMP1-14 (AbsoluteAntibody Inc. Cat # Ab00813-7.1) was determined by mass spectrometry(LC-MS/MS). A single point mutation [D265A] was introduced in the Fcregion of the mouse IgG1 version of RMP1-14 antibody to eliminatebinding to FcgRs, as described in the literature (Nimmerjahn and Ravetch2005 Science 310; 1510-1512¹). Mouse IgG1 [clone MOPC-21], and mouseIgG2a [clone C1.18.4] isotype controls were purchased from BioXCell.PI-7012 and Afuc-PI-7012 (having the CDR sequences of PI37012 andmurinized with a mouse IgG2a format) were produced in Expi293 cells(Thermo Fisher Scientific) or 293/FUT8 knockout cells (University ofToronto) respectively in mouse IgG2a format and purified using MabSelectProtein A resin (GE Life Sciences). The antibodies were eluted with 0.1M citrate buffer (pH 3.0) and buffer exchanged before use.

All experimental procedures involving live animals were approved by theInstitutional Animal Care and Use Committees at Murigenics. 6-8 week oldfemale BALB/c mice were purchased from Taconic and used after one weekof acclimatization to the animal facility. CT26 cells were harvestedwithin 3 to 7 subcultures after thaw from liquid nitrogen stock and thenused for in vivo experiments. Right ventro-lateral area of female Balb/Cmice were shaved and prepared for injection a day in advance. On the dayof tumor inoculation, the cells were harvested and used within 30minutes. To establish subcutaneous tumors, 1×10⁶ CT26 cells wereimplanted and mice were then monitored for tumor growth. Tumor volumeswere calculated from caliper measurements of tumor dimensions using theformula (L×W2)/2, where L is the longer measurement. When tumors reachedan average size of 80-100 cubic mm, the mice were randomized totreatment groups as shown in Table 6:

TABLE 6 Group Treatment Dose/Duration 1 Mouse IgG2a + Mouse IgG1 10mg/Kg + 5 mg/Kg i.p., q5d × 4 2 Mouse IgG2a Anti-PD-1 10 mg/Kg + 5 mg/Kgi.p., q5d × 4 3 Anti-TREM2 [PI-7012] + Mouse IgG1 10 mg/Kg + 5 mg/Kgi.p., q5d × 4 4 Anti-TREM2 [PI-7012] + Anti-PD-1 10 mg/Kg + 5 mg/Kgi.p., q5d × 4 5 Anti-TREM2 [Afuc PI-7012] + 10 mg/Kg + 5 mg/Kg MouseIgG1 i.p., q5d × 4 6 Anti-TREM2 [Afuc PI-7012] + 10 mg/Kg + 5 mg/KgAnti-PD-1 i.p., q5d × 4

Tumor volumes and body weights were monitored twice per week and graphedfor group comparison analyses by one-way ANOVA. Mice were euthanizedwhen tumor volume reached about 2000 cubic mm, when body weights reducedmore than 15% during the study, or for other health related concerns.

Results

We determined whether the affinity of mAb binding to certain FcgR viaglycoengineering (ie, by generating afucosylated versions of theanti-TREM2 mAbs) could increase anti-tumor activity. PI-7012 andafuc-PT-7012 were tested in combination with anti-PD-1 in the CT26 tumormodel. PI-7012 and afuc-PI-7012 displayed similar levels of tumor growthinhibition (79% vs 88% TGI). Treatment with afuc-PI-7012 resulted in a30% cure rate. As seen in FIG. 1A, afuc-PI-7012 had increased anti-tumoractivity when combined with anti-PD-1 than did PI-7012. The impact ofafucosylation of PI-7012 on anti-tumor activity was more clearly seen inthe analysis of the individual mouse tumor volumes (FIGS. 1B and 1C).This demonstrates that afucosylation of anti-TREM2 antibody provides asignificant therapeutic advantage over core-fucosylated antibody.

During the course of the study, there was no significant loss in bodyweight (FIG. 2) in any treatment group. Body weight loss is typicallyused as a surrogate measure for toxicity associated with treatment. Thisdata indicates that short or long-term treatment with anti-TREM2 assingle agent or in combination with anti-PD-1 was well-tolerated andoccurred without any significant toxicity being observed.

Example 5: No Overt Toxicity Associated with Anti-TREM2 TherapyMaterials and Methods

Tissues (lung, liver, brain, kidney, and heart) from mice treated in theabove example were preserved in 10% neutral buffered formalin for atleast 24 hours, processed routinely for histology, cut at 5-6 μm, andsections were stained with hematoxylin and eosin. Stained slides wereexamined using low-power (40-100×) light microscopy, and an image wasobtained through HistoWiz. CD68-positive cells were detected using ananti-CD68 antibody (AbD Serotec) and 8-9 fields of 40× sections werequantified using a light microscope.

Results

Gross morphological analysis by H&E staining of mouse tissues (lung,liver, heart, kidney, and brain) post-treatment did not reveal anymorphological changes in the PI-7012, afuc-PI-7012, and anti-PD-1combination treated mice, compared to isotype control treated mice (FIG.3 shows staining of lung tissue).

In addition to H&E staining, tissues were also stained for macrophagesusing anti-CD68. The intracellular marker CD68 has been used widely inthe literature as a reliable cytochemical marker to immunostainmonocyte/macrophages in inflamed tissues and tumors. In the lung (FIG.4A), as well as in the other tissues analyzed, no discernable change inCD68+ macrophage numbers (FIG. 4B) were observed in any of the treatmentgroups compared to the controls, indicating that anti-TREM2-mediateddepletion occurred specifically in the TME.

Example 6: Limited TREM2 Expression in Healthy Mouse Tissues Materialsand Methods

All animal studies were approved by the Murigenics Animal StudiesCommittee. C57BL/6J-Trem2^(em2Adiuj) (hereafter referred to as TREM2KO)and control C57BL/6J mice were from The Jackson Laboratory. Whole lungs,spleen, and bones were collected and processed immediately for flowcytometry. Blood was collected by cardiac puncture in parallel. Thetissues were processed to single cell suspension using Miltenyi MACStissue dissociation kits. Red blood cells were lysed using 1× red bloodcell lysis buffer (Biolegend). Cells were stained with Fixable ViabilityDye (ThermoFisher Scientific) before processing for cell surfacestaining. Anti-mouse immunophenotyping antibodies were diluted in FACSbuffer (2% FBS, 2 mM EDTA, 1×PBS) along with Fc block and stained for 30minutes on ice. After the staining, the cells were washed twice withFACS buffer and then fixed in 2% paraformaldehyde in PBS for 15 minutes.All data were collected on an LSR Fortessa flow cytometer (BD) or Attuneflow cytometer (Thermo Fisher) and analyzed using FlowJo software.TREM2KO cell staining is shown in the shaded plots, wild type cellstaining is shown in the open plots.

Results

TREM2 is expressed on activated macrophages, immature dendritic cells,osteoclasts, and microglia^(2,3). Cells expressing high levels of TREM2are thought to participate in immune surveillance, cell-cellinteractions, tissue debris clearance, and the resolution of latentinflammatory reactions.⁴ The absence of TREM2 expression on these cellsby gene knockdown or knockout impairs their capacity to phagocytosecellular debris and also increases their production of regulatorycytokines⁵. In a physiological setting, there is very low to nodetectable expression of TREM2 in peripheral blood, spleen, liver, orlung as seen in FACS plots (FIG. 5). However, if lung or liver-residentmacrophages are isolated and stained for TREM2 as pure cellularpopulations, TREM2 expression becomes detectable.

Example 7: TREM2 is Predominantly Expressed on Mouse TAMs Materials andMethods

Tumor tissues were processed to isolate single cell suspension bystandard methods. Briefly, tumors were finely minced with razor bladesand digested in RPMI-1640 medium containing enzymes from Miltenyi MACSdissociation kits. The tumors were processed in GentleMACs as permanufacturer recommendations and incubated at 37 degrees C. forapproximately 40 minutes. The digestion mixture was quenched with PBScontaining 2 mM EDTA and 2% Fetal Bovine Serum. The single cellsuspension was then passed through a 70 um filter and then cells wererinsed with FACS buffer. After centrifugation, the cell pellet wasresuspended in FACS buffer and stained with antibody cocktail toidentify tumor-associated macrophage and other immune cell populations⁶.TREM2KO cell staining is shown in the shaded plots, wild type cellstaining is shown in the open plots.

Results

T cells, B cells, NK cells and other non-myeloid cell populations aswell as CD45-negative cells do not express detectable TREM2 expressionon the cell surface. However, myeloid cell subsets includingtumor-associated macrophages (TAMs) and myeloid derived suppressor cells(MDSCs) express TREM2 to varying degrees on the cell surface. Of thecell types that are positive for TREM2 in the tumor microenvironment,the density of receptor expression on TAMs was significantly higher thanother cell types irrespective of the tumor origin (CT26 and MC38 shownin FIG. 6).

Example 8: Limited TREM2 Expression in Human Peripheral Blood LeukocytesMaterials and Methods

Peripheral blood mononuclear cells (PBMCs) and negatively sorted CD14+monocytes obtained from normal human volunteers were provided byAllCells Inc. The CD14+ monocytes were differentiated in-vitro usingstandard protocol⁵. CD14⁺ monocytes were cultured in complete culturemedium consisting of RPMI-1640 medium supplemented with 2 mML-glutamine, 100 μg per ml streptomycin, 100 U per ml penicillin and 10%heat-inactivated FBS. To trigger differentiation to macrophages, 50ng/mL M-CSF was added to the medium. Medium was supplemented every 2-3days. After 7 days, macrophages were harvested by pipetting and theadherent cells were collected by subsequent trypsinisation. Cells werethen centrifuged and resuspended in RPMI-1640 supplemented withantibiotics, 2% FBS and recombinant human IFN-g and 100 ng/mL LPS. Thesemacrophages were surface stained in parallel with PBMCs using standardmyeloid cocktail to evaluate cell surface staining of TREM2 in cellularsubsets. Cells stained with control mAb are shown in the shaded plots.Cells stained with anti-TREM2 mAb are shown in the open plots.

Results

As seen in FIG. 7, ex-vivo differentiated macrophages displaysignificantly higher cell surface receptor density of TREM2 compared toany PBMC-based cell type evaluated. Similar to observations reported inthe literature, monocytes and some neutrophils express lower levels ofTREM2.

Example 9: TREM2 is Predominantly Expressed on Human TAMs Materials andMethods

Human tumor tissues were obtained from Cooperative Human Tissue Network(CHTN). Fresh human tumor tissues were dissociated into single cellsuspension using Miltenyi MACS dissociation kit and gentleMACS protocol.Single cell suspension of human tumor tissues were surface stained usingpre-validate multi-color FACS panel. All data were collected on an LSRFortessa flow cytometer (BD) or Attune flow cytometer (Thermo Fisher)and analyzed using FlowJo software. Numbers indicate the staining indexfor each population, defined as anti-TREM2 staining minus isotypecontrol staining.

Results

Within the tumor microenvironment, TREM2 expression is differentiallyexpressed to high levels on TAMs (FIG. 8) relative to other cells,making it a translationally relevant marker for TAMs. Representativehistograms of TREM2 antibody (open) or isotype control (shaded) stainingin various cell populations in mucinous adenocarcinoma are shown.Collectively, this data supports the hypothesis that TREM2 targetingagents will aid specific TAM depletion with relatively low to nocollateral impact on peripheral cells or other tissue-resident immunesubsets.

Example 10: Anti-Tumor Efficacy of Anti-TREM2 Antibody in Combinationwith Anti PD-1 in Multiple Syngeneic Tumor Models Materials and Methods

CT26.WT (CRL-2638), Py8119 (CRL-3278), 4T1 (CRL-2539), and EMT6(CTL-2755) cells were purchased from the American Type CultureCollection (ATCC). Panc-02 cells were used at AJES Life Sciences (StonyBrook, N.Y.). Antibodies for in vivo use were all at or below 0.2 EU/mgprotein. The amino acid sequence of the anti-mouse PD-1 antibody fromclone RMP1-14 was determined by mass spectrometry (LC-MS/MS). A singlepoint mutation, D265A, was introduced into the Fc region of the RMP1-14antibody to eliminate binding to FcgRs. Mouse IgG1, clone MOPC-21, andmouse IgG2a, clone C1.18.4, isotype controls were purchased fromBioXCell. PI-7012 and afuc-PI-7012, both as mouse IgG2a, were producedin Expi293 cells (Thermo Fisher Scientific) or 293/FUT8 knockout cells,respectively, and then purified using MabSelect Protein A resin (GE LifeSciences). The mAbs were eluted with 0.1M citrate buffer (pH 3.0) andbuffer exchanged before use.

All experimental procedures involving live animals were approved by theInstitutional Animal Care and Use Committees at Murigenics. FemaleBALB/c or C57BL/6 mice (6-8 weeks old) were purchased from Taconic orThe Jackson Laboratory and used after one week of acclimatization at theanimal facility. Tumor cells were harvested within 3 to 7 subculturesafter thaw from liquid nitrogen stock and then used for the in vivoexperiments. The right ventro-lateral area of female mice were shavedand prepared for injection a day in advance of tumor cell inoculation.On the day of tumor inoculation, the cells were harvested and usedwithin 30 minutes. To establish subcutaneous tumors, 1×10⁶ CT26, EMT6,or Panc-02 cells, or 1×10⁵ 4T1 cells were implanted into appropriatestrains of mice, and then the animals were monitored for tumor growth.Equal volumes of single cell suspension of Py8119 cells were mixed withMatrigel (Corning Cat #354248 or 354263) before implanting 2×10⁶ cellsper mouse.

Tumor volumes were calculated using caliper measurements of tumordimensions using the formula (L×W2)/2, where L is the longermeasurement. When tumors reached an average size of 80-100 cubic mm, themice were randomized to treatment groups as shown in Table 7.

Tumor volumes and body weights were monitored twice a week and graphedfor group comparison analyses by one-way ANOVA. Mice were euthanizedwhen the tumor volumes reached 2,000 cubic mm, or when body weights werereduced more than 15% during the study.

TABLE 7 Group Treatment Dose/D oration 1 Mouse IgG1 + Mouse IgG2a 5mg/kg + 15 mg/kg i.p., q5d × 4 2 Anti-PD-1 + Mouse IgG2a 5 mg/kg + 15mg/kg i.p., q5d × 4 3 Mouse IgG1 + Anti-TREM2 5 mg/kg + 15 mg/kg i.p.,q5d × 4 4 Anti-PD-1 + Anti-TREM2 5 mg/kg + 15 mg/kg i.p., q5d × 4

Results

The results are summarized in Table 8. Tumor growth inhibition (% TGI)was determined at the end of the dosing period (t) by the formula: %TGI=(1−{Tt/T0/Ct/C0}/1−{C0/Ct})×100 where Tt=median tumor volume ofcombination-treated at time t, T0=median tumor volume ofcombination-treated at time 0, Ct=median tumor volume of isotype controlat time t and C0=median tumor volume of isotype-treated at time 0(before start of treatment).

TABLE 8 Model Strain Activity CT26 [CRC] BALB/c ~50-85% TGI in CT26 upontreatment in combination with anti- PD-1, as well as 40-60% CompleteResponse Py8119 [TNBC] C57BL/6 ~56% TGI in combination with anti-PD-14T1 [TNBC] BALB/c ~23% TGI in combination with anti-PD-1 EMT6 [Mammary]BALB/c ~63% TGI in combination with anti-PD-1 and 20% Complete ResponsePane02 [Pancreatic] C57BL/6 ~63% TGI in combination with anti-PD-1

FIG. 9A-F show the anti-tumor activity of anti-TREM2 PI-7012 orafuc-PI7012 in combination with anti-PD-1 in multiple syngeneic mousetumor models. Anti-TREM2 mAb afuc-PI7012 combined with anti-PD-1 mAbresults in significant anti-tumor activity in the Panc-02 pancreatictumor model. FIG. 9A shows the mean+/−standard deviation of the averagetumor volumes of 10 mice in each group. FIGS. 9B, 9C, 9D, and 9E showthe tumor volumes from individual animals in each treatment group overtime. FIG. 9F shows the statistical analysis of the group average tumorvolumes on day 32 after implant. Differences in tumor volumes betweengroups were evaluated using the statistical analyses available in theGraph Pad Prism software. One-way ANOVA followed by Sidak's multiplecomparison test was performed on the study data.

As seen in FIGS. 9A and 9D, the subcutaneous Panc-02 tumor is notresponsive to an anti PD-1 mAb single agent immune checkpoint blockadetherapy, or to anti-TREM2 mAb afuc-PI-7012 therapy alone. However, thecombination treatment of Panc-02 tumor bearing animals with anti-TREM2mAb afuc-PI-7012 and anti-PD-1 mAbs resulted in significant tumor growthinhibition.

The combination therapeutic strategy of myeloid-tuning along with immunecheckpoint-mediated reversal of CD8 T-cell exhaustion was tested inmultiple syngeneic tumor models. As shown in Table 8, the combination ofanti-TREM2 and anti-PD-1 mAbs resulted in significant tumor growthinhibition, as well as complete regression in several of the tumormodels tested. It is important to note that these syngeneic models weregrown in two different mouse strain backgrounds (prototypical Th-1C57BL/6 and Th-2 BALB/c strains) which are known to have significantdifferences in the composition of the immune infiltrates in tumors grownin these strains in vivo.

Example 11: Long-Term, Anti-Tumor Immune Memory is Elicited in MiceResponding to Anti-TREM2 mAb Plus Anti PD-1 mAb Combination TreatmentMaterials and Methods

BALB/c mice that were tumor-free from previous studies after theanti-TREM2 mAb plus anti-PD-1 mAb treatment described in Example 9 werere-challenged three months later with 1×10⁶CT26 tumor cells. Tumorvolume was measured for 25 days post implant. Age-matched treatmentnaïve mice received equivalent number of CT26 cells and tracked fortumor growth during the study period. No additional treatment wasprovided to the mice during the study period.

Results

Mice that were cured of their CT26 tumors following treatment with thecombination of anti-TREM2 mAb afuc-PI-7012 and anti-PD-1 mAb establishedan effective anti-tumor memory response (FIG. 10). Cured mice were ableto reject any new tumor growth even in the absence of additionaltherapy, indicating long-term immune memory against the original,implanted tumor. This form of long-term immune memory utilizesmaintenance of a vigorous CD8+ effector memory response.

REFERENCES

-   1. Nimmerjahn F. & Ravetch, J. V. Divergent Immunoglobulin G    Subclass Activity Through Selective Fc Receptor Binding. Science    (80-.). 310, 1510 LP-1512 (2005).-   2. Ford, J. W. & McVicar, D. W. TREM and TREM-like receptors in    inflammation and disease. Curr. Opin. Immunol. 21, 38-46 (2009).-   3. Colonna, M. TREMs in the immune system and beyond. Nat. Rev.    Immunol. 3, 445 (2003).-   4. Takahashi, K., Rochford, C. D. P. & Neumann, H. Clearance of    apoptotic neurons without inflammation by microglial triggering    receptor expressed on myeloid cells-2. J. Exp. Med. 201, 647 LP-657    (2005).-   5. Piccio, L. et al. Blockade of TREM-2 exacerbates experimental    autoimmune encephalomyelitis. Eur. J. Immunol. 37, 1290-1301 (2007).-   6. Broz, M. L. et al. Dissecting the Tumor Myeloid Compartment    Reveals Rare Activating Antigen-Presenting Cells Critical for T Cell    Immunity. Cancer Cell 26, 638-652 (2017).

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

Sequences SEQ ID NO Name Sequence  1 37012_VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREWAGSGYFDY WGQGTLVTVSS  237012_VL DIQMTQSPSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNRFTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIK  3 37013_VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREWAGSGYFDY WGQGTLVTVSS  437013_VL DIQMTQSPSSLSASVGDRVTMTCKASQNVGNNLAWYQQKPGKAPKLLLYYTSNRFTGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCQRIYNSPWTFGQGTKLELK  5 37014_VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVASLTNSGGSTYYADSVKGRFTLSRDNSKNTLYLQMNSLRAEDTAVYYCTREWAGSGYFDY WGQGTLVTVSS  637014_VL DIQMTQSPSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNRFTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIK  7 37017_VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEWAGSGYFDY WGQGTLVTVSS  837017_VL DIQMTQSPSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNRFTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIK  9 CDR-H1 FSNYYMA10 CDR-H2 SLTNSGGSTY 11 CDR-H3 EWAGSGY 12 CDR-L1 NVGNNLA 13 CDR-L2YTSNRFT 14 CDR-L3 RIYNSPW 15 TREM2MEPLRLLILLFVTELSGAHNTTVFQGVAGQSLQVSCPYDSMKHWGRRKAWCRQL HumanGEKGPCQRVVSTHNLWLLSFLRRWNGSTAITDDTLGGTLTITLRNLQPHDAGLY ProteinQCQSLHGSEADTLRKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEHSISRSLLEGEIPFPPTSILLLLACIFLIKILAASALWAAAWHGQKPGTHPPSELDCGHD PGYQLQTLPGLRDT 16TREM2 ATGGAGCCTCTCCGGCTGCTCATCTTACTCTTTGTCACAGAGCTGTCCGGAGCC NucleotideCACAACACCACAGTGTTCCAGGGCGTGGCGGGCCAGTCCCTGCAGGTGTCTTGC (CDS)CCCTATGACTCCATGAAGCACTGGGGGAGGCGCAAGGCCTGGTGCCGCCAGCTGGGAGAGAAGGGCCCATGCCAGCGTGTGGTCAGCACGCACAACTTGTGGCTGCTGTCCTTCCTGAGGAGGTGGAATGGGAGCACAGCCATCACAGACGATACCCTGGGTGGCACTCTCACCATTACGCTGCGGAATCTACAACCCCATGATGCGGGTCTCTACCAGTGCCAGAGCCTCCATGGCAGTGAGGCTGACACCCTCAGGAAGGTCCTGGTGGAGGTGCTGGCAGACCCCCTGGATCACCGGGATGCTCGAGATCTCTGGTTCCCCGGGGAGTCTGAGAGCTTCGAGGATGCCCATGTGGAGCACAGCATCTCCAGGAGCCTCTTGGAAGGAGAAATCCCCTTCCCACCCACTTCCATCCTTCTCCTCCTGGCCTGCATCTTTCTCATCAAGATTCTAGCAGCCAGCGCCCTCTGGGCTGCAGCCTGGCATGGACAGAAGCCAGGGACACATCCACCCAGTGAACTGGACTGTGGCCATGACCCAGGGTATCAGCTCCAAACTCTGCCAGGGCTGAGAGACACGTGA 17 TREM2MGPLHQFLLLLITALSQALNTTVLQGMAGQSLRVSCTYDALKHWGRRKAWCRQL MouseGEEGPCQRVVSTHGVWLLAFLKKRNGSTVIADDTLAGTVTITLKNLQAGDAGLY ProteinQCQSLRGREAEVLQKVLVEVLEDPLDDQDAGDLWVPEESSSFEGAQVEHSTSRNQETSFPPTSILLLLACVLLSKFLAASILWAVARGRQKPGTPVVRGLDCGQDAGH QLQILTGPGGT 18Frame H1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNS GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEWAGSGYFDYWGQGTL VTVSS 19 Frame H2EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNS GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREWAGSGYFDYWGQGTL VTVSS 20 Frame H3EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVASLTNS GGSTYYADSVKGRFTLSRDNSKNTLYLQMNSLRAEDTAVYYCTREWAGSGYFDYWGQGTL VTVSS 21 3-23*01EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGS Frame VH GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGQGTLVTVSS 22 Frame L1DIQMTQSPSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNRFTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIK 23 Frame L2DIQMTQSPSSLSASVGDRVTMTCKASQNVGNNLAWYQQKPGKAPKLLLYYTSNRFTGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCQRIYNSPWTFGQGTKLELK 24 3-23*01DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSL Frame VLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPFGQGTKLEIK 25 FullEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNS 37012_HGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREWAGSGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK26 Full DIQMTQSPSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNR 37012_LFTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 27 FullEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNS 37013_HGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREWAGSGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK28 Full DIQMTQSPSSLSASVGDRVTMTCKASQNVGNNLAWYQQKPGKAPKLLLYYTSNR 37013_LFTGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCQRIYNSPWTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 29 FullEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVASLTNS 37014_HGGSTYYADSVKGRFTLSRDNSKNTLYLQMNSLRAEDTAVYYCTREWAGSGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK30 Full DIQMTQSPSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNR 37014_LFTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 31 FullEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMAWVRQAPGKGLEWVSSLTNS 37017_HGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEWAGSGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK32 Full DIQMTQSPSSLSASVGDRVTITCKASQNVGNNLAWYQQKPGKAPKLLIYYTSNR 37017_LFTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQRIYNSPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 33 RatEVQLVESGGG LVQPGRSLKL SCAASGFTFS NYYMAWVRQA PTKGLEWVAS IgG2BLTNSGGSTYY RDSVKGRFTL SRDNAKSTLY LQMDSLRSED TATYYCTREW clone #AGSGYFDYWG QGVMVTVSSA QTTAPSVYPL APGCGDTTSS TVTLGCLVKG 237920YFPEPVTVTW NSGALSSDVH TFPAVLQSGL YTLTSSVTSS TWPSQTVTCN heavyVAHPASSTKV DKKVERRDGG IGHKCPTCPT CHKCPVPELL GGPSVFLFPP chainKPKDILLLSQ NAKVTCVVVD VSEEEPDVQF SWFVNNVEVH TAQTQPREEQYNSTFRVVSA LPLQHQDWMS GKEFKCKVNN KALPSPIEKT LSKPKGLVRKPQVYVMGPPT EQLTEQTVSL TCLTSGFLPN DIGVEWTSNG HIEKNYKNTEPVMDSDGSFF MYSKLNVERS RWDSRAPFVC SVVHEGLHNH HVEKSLSRPP G 34 RatNIVMTQSPKS MSLSVGDRVT MNCKASQNVG NNLAWYQQKP GQSPKLLLYY IgG2BTSNRFTGVPD RFTGGGYGTD FTLTINSVQA EDAAFYYCQR IYNSPWTFGG clone #GTKLELKRAD AAPTVSIFPP STEQLATGGA SVVCLMNNFY PRDISVKWKI 237920DGTERRDGVL DSVTDQDSKD STYSMSSTLS LTKADYESHN LYTCEVVHKT lightSSSPVVKSFN RNEC chain

1.-95. (canceled)
 96. An isolated antibody that binds to human TREM2(SEQ ID NO: 15), wherein the antibody i) competes for binding to mouseTREM2 (SEQ ID NO: 17) with the 37017 antibody (SEQ ID NOs: 31 and 32);and ii) comprises a human Fc region.
 97. The isolated antibody of claim96, wherein the antibody comprises a heavy chain comprising a variableheavy (VH) chain sequence comprising three heavy chain CDR sequences,CDR-H1, CDR-H2, and CDR-H3, and a light chain comprising a variablelight (VL) chain sequence comprising three light chain CDR sequences,CDR-L1, CDR-L2, and CDR-L3, wherein: a. CDR-H3 comprises the sequenceset forth in SEQ ID NO: 11, and b. CDR-L3 comprises the sequence setforth in SEQ ID NO:
 14. 98. The isolated antibody of claim 97, whereinthe antibody comprises a heavy chain comprising a variable heavy (VH)chain sequence comprising three heavy chain CDR sequences, CDR-H1,CDR-H2, and CDR-H3, and a light chain comprising a variable light (VL)chain sequence comprising three light chain CDR sequences, CDR-L1,CDR-L2, and CDR-L3, wherein: a. CDR-H1 comprises the sequence set forthin SEQ ID NO: 9, b. CDR-H2 comprises the sequence set forth in SEQ IDNO: 10, c. CDR-H3 comprises the sequence set forth in SEQ ID NO: 11, d.CDR-L1 comprises the sequence set forth in SEQ ID NO: 12, e. CDR-L2comprises the sequence set forth in SEQ ID NO: 13, and f. CDR-L3comprises the sequence set forth in SEQ ID NO:
 14. 99. The isolatedantibody of claim 98, wherein the VH chain sequence comprises the VHsequence shown in SEQ ID NO: 1; and the VL chain sequence comprises theVL sequence shown in SEQ ID NO:
 2. 100. The isolated antibody of claim98, wherein the VH chain sequence comprises the VH sequence shown in SEQID NO: 1, 3, or 5 and the VL chain sequence comprises the VL sequenceshown in SEQ ID NO: 2, 4, or
 6. 101. The isolated antibody of claim 98,wherein the heavy chain comprises the heavy chain sequence shown in SEQID NO: 25 and the light chain comprises light chain sequence shown inSEQ ID NO:
 26. 102. The isolated antibody of claim 98, wherein theantibody is afucosylated, and wherein the VH chain sequence comprisesthe VH sequence shown in SEQ ID NO: 1; and the VL chain sequencecomprises the VL sequence shown in SEQ ID NO:
 2. 103. The isolatedantibody of claim 98, wherein the antibody is afucosylated, and whereinthe VH chain sequence comprises the VH sequence shown in SEQ ID NO: 1,3, or 5 and the VL chain sequence comprises the VL sequence shown in SEQID NO: 2, 4, or
 6. 104. The isolated antibody of claim 98, wherein theantibody is afucosylated, and wherein the heavy chain comprises theheavy chain sequence shown in SEQ ID NO: 25 and the light chaincomprises light chain sequence shown in SEQ ID NO:
 26. 105. The isolatedantibody of claim 98, wherein the antibody binds to human TREM2 with aKD of less than or equal to about 1, 2, 3, 4, or 5×10-9 M, as measuredby surface plasmon resonance (SPR) assay.
 106. The isolated antibody ofclaim 98, wherein the antibody is afucosylated.
 107. The isolatedantibody of claim 98, wherein the human Fc region is wild-type humanIgG1 Fc.
 108. The isolated antibody of claim 98, wherein the antibody isafucosylated and the human Fc region is wild-type human IgG1 Fc. 109.The isolated antibody of claim 98, wherein the antibody is capable ofspecifically killing, depleting, or disabling TREM2+ myeloid cells, andwherein the antibody has at least one of antibody-dependentcell-mediated cytotoxicity (ADCC) activity, antibody-mediated cellularphagocytosis (ADCP) activity, and complement-dependent cytotoxicity(CDC) activity.
 110. A pharmaceutical composition comprising theantibody of claim 96 and a pharmaceutically acceptable excipient.
 111. Amethod of producing the antibody of claim 96 comprising expressing theantibody in a host cell and isolating the expressed antibody.
 112. Amethod of treating cancer in a subject in need thereof, comprisingadministering to the subject an isolated antibody that binds to humanTREM2 (SEQ ID NO: 15), wherein the antibody i) competes for binding tomouse TREM2 (SEQ ID NO: 17) with the 37017 antibody (SEQ ID NOs: 31 and32); and ii) comprises a human Fc region.
 113. The method of claim 112,wherein the antibody comprises a heavy chain comprising a variable heavy(VH) chain sequence comprising three heavy chain CDR sequences, CDR-H1,CDR-H2, and CDR-H3, and a light chain comprising a variable light (VL)chain sequence comprising three light chain CDR sequences, CDR-L1,CDR-L2, and CDR-L3, wherein: a. CDR-H1 comprises the sequence set forthin SEQ ID NO: 9, b. CDR-H2 comprises the sequence set forth in SEQ IDNO: 10, c. CDR-H3 comprises the sequence set forth in SEQ ID NO: 11, d.CDR-L1 comprises the sequence set forth in SEQ ID NO: 12, e. CDR-L2comprises the sequence set forth in SEQ ID NO: 13, and f. CDR-L3comprises the sequence set forth in SEQ ID NO:
 14. 114. The method ofclaim 112, wherein the subject has previously received, is concurrentlyreceiving, or will subsequently receive an immunotherapy, wherein theimmunotherapy is at least one of: a checkpoint inhibitor; a checkpointinhibitor of T cells; anti-PD1 antibody; anti-PDL1 antibody; anti-CTLA4antibody; adoptive T cell therapy; CAR-T cell therapy; a dendritic cellvaccine; a monocyte vaccine; an antigen binding protein that binds botha T cell and an antigen presenting cell; a BiTE dual antigen bindingprotein; a toll-like receptor ligand; a cytokine; a cytotoxic therapy; achemotherapy; a radiotherapy; a small molecule inhibitor; a smallmolecule agonist; an immunomodulator; and an epigenetic modulator. 115.The method of claim 114, wherein the immunotherapy is an anti-PD1antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody.
 116. Themethod of claim 112, wherein the cancer is a solid cancer or a liquidcancer.
 117. The method of claim 112, wherein the cancer is selectedfrom the group consisting of: colon, breast, melanoma, kidney,hepatobiliary, head-neck squamous carcinoma (HNSC), pancreatic, bladder,glioblastoma, prostate, lung, ovarian, gastric, kidney, bladder,esophageal, renal, melanoma, and mesothelioma.
 118. A method of killing,disabling, or depleting TREM2+ myeloid cells of a subject, comprisingadministering to the subject an isolated antibody that binds to humanTREM2 (SEQ ID NO: 15), wherein the antibody i) competes for binding tomouse TREM2 (SEQ ID NO:17) with the 37017 antibody (SEQ ID NOs: 31 and32); and ii) comprises a human Fc region.
 119. The method of claim 118,comprising contacting the myeloid cells with an antibody comprising ahuman IgG1 Fc, a heavy chain comprising a variable heavy (VH) chainsequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, andCDR-H3, and a light chain comprising a variable light (VL) chainsequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, andCDR-L3, wherein: a. CDR-H1 comprises the sequence set forth in SEQ IDNO: 9, b. CDR-H2 comprises the sequence set forth in SEQ ID NO: 10, c.CDR-H3 comprises the sequence set forth in SEQ ID NO: 11, d. CDR-L1comprises the sequence set forth in SEQ ID NO: 12, e. CDR-L2 comprisesthe sequence set forth in SEQ ID NO: 13, and f. CDR-L3 comprises thesequence set forth in SEQ ID NO:
 14. 120. The method of claim 119,wherein the antibody has at least one of antibody-dependentcell-mediated cytotoxicity (ADCC) activity, complement-dependentcytotoxicity (CDC) activity, and antibody-mediated phagocytosis (ADCP)activity.
 121. The method of claim 119, wherein the TREM2+ myeloid cellscomprise at least one of dendritic cells, tumor-associated macrophages(TAMs), neutrophils, or monocytes.
 122. The method of claim 119, whereinthe subject has a tumor and the TREM2+ myeloid cells are intratumoral.123. The method of claim 119, wherein the subject has previouslyreceived, is concurrently receiving, or will subsequently receive animmunotherapy.
 124. The method of claim 119, wherein the immunotherapyis at least one of: a checkpoint inhibitor; a checkpoint inhibitor of Tcells; anti-PD1 antibody; anti-PDL1 antibody; anti-CTLA4 antibody;adoptive T cell therapy; CAR-T cell therapy; a dendritic cell vaccine; amonocyte vaccine; an antigen binding protein that binds both a T celland an antigen presenting cell; a BiTE dual antigen binding protein; atoll-like receptor ligand; a cytokine; a cytotoxic therapy; achemotherapy; a radiotherapy; a small molecule inhibitor; a smallmolecule agonist; an immunomodulator; and an epigenetic modulator. 125.The method of claim 119, wherein the immunotherapy is an anti-PD1antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody.